Crp capture/detection of gram positive bacteria

ABSTRACT

Described herein are engineered microbe-targeting molecules, microbe-targeting articles, kits comprising the same, and uses thereof. Such microbe-targeting molecules, microbe-targeting articles, or the kits comprising the same can bind or capture of a microbe or microbial matter thereof, and can thus be used in various applications, such as diagnosis or treatment of an infection caused by microbes in a subject or any environmental surface.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 16/669,805 filed Oct.31, 2019, which is a divisional of U.S. Ser. No. 15/105,298 filed Jun.16, 2016, which issued as U.S. Pat. No. 10,513,546 on Dec. 24, 2019,which is a 371 National Phase Entry of International Patent ApplicationNo. PCT/US2014/071293 filed on Dec. 18, 2014 which claims benefit under35 U.S.C. § 119(e) of the U.S. Provisional Application No. 61/917,705,filed Dec. 18, 2013, the contents of which are incorporated herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.N66001-11-1-4180 awarded by Defense Advanced Research Projects Agency(DARPA). The government has certain rights in the invention.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “002806-08011-PCT_SL”, creation date of Jun. 15, 2016 and asize of 88,383 bytes. The sequence listing submitted via EFS-Web is partof the specification and is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

Described herein relates generally to molecules, products, kits andmethods for detecting and/or removing microbes in a sample or a targetarea, including bodily fluids such as blood and tissues of a subject,food, water, and environmental surfaces.

BACKGROUND

Sepsis is a major cause of morbidity and mortality in humans and otheranimals. In the United States, sepsis is the second leading cause ofdeath in intensive care units among patients with non-traumaticillnesses. It is also the leading cause of death in young livestock,affecting 7.5-29% of neonatal calves, and is a common medical problem inneonatal foals. Despite the major advances of the past several decadesin the treatment of serious infections, the incidence and mortality dueto sepsis continues to rise.

Sepsis results from the systemic invasion of microorganisms into bloodand can present two distinct problems. First, the growth of themicroorganisms can directly damage tissues, organs, and vascularfunction. Second, toxic components of the microorganisms can lead torapid systemic inflammatory responses that can quickly damage vitalorgans and lead to circulatory collapse (i.e., septic shock) and, oftentimes, death.

There are three major types of sepsis characterized by the type ofinfecting organism. For example, gram-negative sepsis is the mostfrequently isolated (with a case fatality rate of about 35%). Themajority of these infections are caused by Escherichia coli, Klebsiellapneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such asthe Staphylococci and Streptococci are the second major cause of sepsis.The third major group includes fungi, with fungal infections causing arelatively small percentage of sepsis cases, but with a high mortalityrate; these types of infections also have a higher incidence inimmunocomprised patients.

Many patients with septicemia or suspected septicemia exhibit a rapiddecline over a 24-48 hour period. It has been reported that patientswith septic shock require adapted treatment in less than 6 hours inorder to benefit from antimicrobial therapy. Thus, rapid and reliablediagnostic and treatment methods are essential for effective patientcare. Unfortunately, a confirmed diagnosis as to the type of infection,e.g., sepsis, traditionally requires microbiological analysis involvinginoculation of blood cultures, incubation for 18-24 hours, plating thecausative microorganism on solid media, another incubation period, andfinal identification 1-2 days later. Even with immediate and aggressivetreatment, some patients can develop multiple organ dysfunction syndromeand eventually death. Hence, there remains a strong need for improvedtechniques for diagnosis and treatment of patients with infectiousdiseases, blood-borne infections, sepsis, or systemic inflammatoryresponse syndrome. The ability to rapidly detect infectious pathogens infood, water, and/or environmental surfaces would also have great valuefor preventing infections and sepsis in the population.

SUMMARY

Embodiments described herein are based on, at least in part, engineeringa microbe-targeting molecule or a microbe-binding molecule. The terms“microbe-targeting molecule” and “microbe-binding molecule.” Theengineered microbe-targeting molecules described herein provide avaluable building block for various applications including, but notlimited to, diagnosis or treatment of diseases caused by microbes orpathogens, removal of microbes or pathogens from a sample, includingbodily fluids and tissues of a subject, foods, water, or anenvironmental surface; and development of targeted drug deliverydevices.

Generally, the microbe-targeting moleculecomprise at least one firstdomain comprising at least a portion of a C-reactive protein (CRP) andat least one second domain. The first and second domains are conjugatedtogether via a linker. In some embodiments, the second domain can beselected from the group consisting of Fc region of an immunoglobulin;microbe-binding domain of a microbe-binding protein, wherein themicrobe-binding protein is not CRP; neck region of a lectin; adetectable label; domain for conjugation to surface of a carrierscaffold; at least a portion of a C-reactive protein; and anycombinations thereof.

The engineered microbe-targeting molecules described herein can be usedas soluble proteins, e.g., in therapeutic compositions, or beimmobilized to a carrier scaffold for various applications ranging fromdiagnosis and/or treatment of a microbial infection or disease, tomicrobe-clearing compositions or devices, to drug delivery. A carrierscaffold comprising a microbe-targeting molecule conjugated therewith isalso referred to as a microbe-targeting article herein.

The microbe-targeting molecules can be used to capture, detect, orremove microbes or pathogens in a sample, e.g., blood and tissues.Accordingly, the microbe-targeting molecules disclosed herein can beused to in assays for detecting the presence or absence of, and/ordifferentiating between, different microbes or pathogens in a testsample or environmental surfaces. Detection assay can comprise anenzyme-linked immunosorbent assay (ELISA), fluorescent linkedimmunosorbent assay (FLISA), immunofluorescent microscopy, fluorescencein situ hybridization (FISH), electrochemical sensor assay, or any otherradiological, chemical, enzymatic or optical detection assays. Further,the engineered microbe-targeting molecules disclosed herein can beformulated as an antibiotic or antiseptic for use in variousapplications, e.g., wound dressings, alone or in combination with otherwound dressing protocols, e.g., silver nanoparticles and other woundtreatment.

The disclosure also provides kits and assays for detecting the presenceor absence of microbes, and/or differentiating between, differentmicrobes or pathogens in a test sample or an environmental surface. Suchkits can be used for analysis, e.g., by an enzyme-linked immunosorbentassay (ELISA), fluorescent linked immunosorbent assay (FLISA),immunofluorescent microscopy, fluorescence in situ hybridization (FISH),or any other radiological, chemical, enzymatic or optical detectionassays. In some embodiments, the kits and assays described herein can beadapted for antibiotic susceptibility tests, e.g., to determinesusceptibility of a microbe in a test sample to one or more antibiotics,regardless of whether the identity of the microbe is known or not.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic of an exemplary microbial capture/detectionprocess or diagnosis process.

FIG. 2 is a schematic diagram of an exemplary ELISA assay comprisingengineered microbe-targeting magnetic microbeads according to one ormore embodiments. The ELISA assay can be used for any diagnosticapplications, e.g., for sepsis tests.

FIG. 3 is a schematic diagram showing one or more embodiments of adipstick assay for microbial detection. The microbe-targeting moleculecan be attached to a membrane (for example Biodyne membrane). Themembrane can be mixed with a test sample (e.g., blood sample), washed,incubated with a desired detecting or lableling molecule (e.g.,enzyme-linked microbe-targeting molecule or specific antibody forcertain microbes, e.g., bacteria or fungus), washed and added with areadout reagent for colorimetric development. The dipstick assay can beperformed manually or modified for automation.

FIG. 4 is a schematic diagram showing one or more embodiments of anELISA-based test for microbial detection. A test sample (e.g., bloodsample) can be added into a single tube (e.g., a blood collectioncontainer such as EDTA VACUTAINER®) containing lyophilizedmicrobe-targeting molecule coated magnetic particles. The ELISA-basedtest can be performed manually or modified for automation. In someembodiments, the single-tube based ELISA assay can be used to detectmicrobes or pathogens.

FIG. 5A-FIG. 5B shows an example of CRP-HRP ELISA: Detection ofEnterococcus faecalis (FIG. 5A) and NOT E. coli (FIG. 5B) from wholeblood. E. faecalis and E. coli were grown to 0.5 McFarlan in RPMI 5%Glucose, serial dilutions made, spiked into whole blood, used forcapture and assayed by CRP-HRP ELISA.

FIG. 6 is a schematic representation showing C-terminus ofmicrobe-targeting molecule (CRP-X-Fc, X is a linker) according to anembodiment of the invention blocks histadine 38 of the CRP.

FIG. 7 is a schematic representation showing C1q component binding andcomplement activation is on A face of CRP and CRP ligand binding, e.g.,microbe binding, is on B face of CRP.

FIG. 8 is a schematic representation showing Fc in CRP-Fc is bound to Aface of CRP thereby inhbiting C1q binding and complement activation.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are microbe-targeting molecules, compositionscomprising the same, processes or assays, and kits for detecting thepresence or absence of a microbe in a test sample. The microbe-targetingmoleucles disclosed herein can also be used for separating microbes froma test sample in vivo, in situ or in vitro. Generally, themicrobe-targeting molecules disclosed herein can bind with or capture atleast one microbe. The microbe can be an intact or whole microbe or anymatter or component that is derived, originated or secreted from amicrobe. Any matter or component that is derived, originated or secretedfrom a microbe is also referred to as “microbial matter” herein. Thus,the microbe-targeting molecules disclosed herein can bind/capture anintact or whole microbe or microbial matter derived, originated orsecreted from the microbe. Exemplary microbial matter that can bind tothe microbe-targeting molecule can include, but is not limited to, acell wall component, an outer membrane, a plasma membrane, a ribosome, amicrobial capsule, a pili or flagella, any fragments of theaforementioned microbial components, any nucleic acid (e.g., DNA,including 16S ribosomal DNA, and RNA) derived from a microbe, microbialendotoxin (e.g., lipopolysaccharide), and the like. In addition,microbial matter can encompass non-viable microbial matter that cancause an adverse effect (e.g., toxicity) to a host or an environment.The terms “microbe-binding molecule(s)” and “microbe-targetingmolecule(s)” are used interchangeably herein.

Various aspects disclosed herein are based on inventors' discovery thatC-reactive protein (CRP) can bind with gram-positive microbe and can beused for capturing/detecting microbes. To the inventors' knowledge, thisis the first use of CRP as a pathogen captured/detector and is differentfrom the current use of CRP as a biomarker. While the prior art uses CRPas a biomarker, the inventors have discovered inter alia that CRP canused to capture/detect microbes. Further, CRP binds gram positiveorganisms (such as Strep agalactiae (AKA GBS and Strep pneumonia) thatbind poorly with currently known engineered microbe-binding moleucles.Gram positive organisms like are responsible for lung and URTIinfections. Accordingly, the molecules, compositions, assays, andmethods disclosed herein can be used in diagnosis, imagining andtreatment of infections with gram-positive microbes.

In accordance with the various embodiments described herein, themicrobe-targeting molecules comprise at least one first domaincomprising at least a portion of a C-reactive protein (CRP) and at leastone second domain. The first and second domains are conjugated togethervia a linker.

It is noted that the first domain and second domain in themicrobe-targeting molecule can be present in any order. For example, thefirst domain can be first followed by the second domain, or the seconddomain can be first followed by the first domain.

Without limitations, the first domain can comprise the full length CRPor a fragment thereof retaining microbe binding activity. In addition tothe CRP amino acid sequence, the first domain can further comprise oneor more amino acids (e.g., one, two, three, four, five, six, seven,eight, nine, ten, or more) amino acids on the N- or C-terminus of theCRP sequence. Generally, the first domain can have an amino acidsequence of about 10 to about 300 amino acid residues. In someembodiments, the first domain can have an amino acid sequence of about50 to about 250 amino acid residues. In some embodiments, the microbesurface-binding domain can have an amino acid sequence of at least about5, at least about 10, at least about 15, at least about 20, at leastabout 30, at least about 40, at least about 50, at least about 60, atleast about 70, at least about 80, at least about 90, at least about 100amino acid residues or more. For any known sequences of CRP one of skillin the art can determine the optimum length of amino acid sequence forretaining microbe-binding activity.

Without limitations, the CRP can be from any source available to one ofskill in the art. For example, the CRP can be from a mammalian source.For example, the CRP can be human CRP (NCBI Reference Sequence:NP_000558.2, SEQ ID NO: 1) or mouse CRP (NCBI Reference Sequence:NP_031794.3, SEQ ID NO: 2). In some embodiments, the first domaincomprises an amino acid sequence comprising amino acids 19-224 of thehuman. In some embodiments, the first domain comprises the amino acidsequence SEQ ID NO: 3 or SEQ ID No: 4. In some embodiments, the firstdomain comprises amino acid sequence SEQ ID NO: 39.

Modifications to the first domain, e.g., by conservative substitution,are also within the scope described herein. In some embodiments, the CRPor a fragment thereof used in the microbe-targeting molecules describedherein can be a wild-type molecule or a recombinant molecule.

In some embodiments, 100% of the first domain can be used to bind tomicrobes or pathogens. In other embodiments, the first domain cancomprise additional regions that are not capable of binding to amicrobe, but can have other characteristics or perform other functions,e.g., to provide flexibility to the first domain when interacting withmicrobes or pathogens. In some embodiments, the first domain cancomprise a peptidomimetic that mimics CRP or a fragment thereof that canbind to the surface of a microbe or pathogen, or microbial matter.

The second domain can be selected to provide a desired function orproperty to the microbe-binding molecules disclosed herein. For example,the second domain can be selected or configured according to a specificneed or use of the microbe-binding molecule. By way of example only, insome embodiments, second domain can be selected or configured to have asufficient length and flexibility such that it can allow for the firstdomain to orient in a desired orientation with respect to a microbe. Insome embodiments, the second domain can be selected or configured toallow multimerization of at least two engineered microbe-targetingmolecules (e.g., to from a di-, tri-, tetra-, penta-, hexa- or highermultimeric complex) while retaining biological activity (e.g.,microbe-binding activity). In some embodiments, the second domain can beselected or configured to interact with the linker to allowmultimerization of at least two engineered microbe-targeting molecules(e.g., to from a di-, tri-, tetra-, penta-, hexa- or higher multimericcomplex) while retaining microbe-binding activity.

In some embodiments, the second domain can be selected or configured tofacilitate expression and purification of the engineeredmicrobe-targeting molecule described herein. In some embodiments, thesecond domain can be selected or configured to provide a recognitionsite for a protease or a nuclease. In addition, the second domain can benon-reactive with the functional components of the engineered moleculedescribed herein. For example, minimal hydrophobic or charged characterto react with the first domain.

In some embodiments, the second domain can include at least a portion ofan immunoglobulin, e.g., IgA, IgD, IgE, IgG and IgM including theirsubclasses (e.g., IgG1), or a modified molecule or recombinant thereof.In some embodiments, the second domain can comprise a portion offragment crystallization (Fc) region of an immunoglobulin or a modifiedversion thereof. In such embodiments, the portion of the Fc region thatcan be comprised in the second domain can comprise at least one regionselected from the group consisting of a hinge region, a CH2 region, aCH3 region, and any combinations thereof. By way of example, in someembodiments, a CH2 region can be excluded from the portion of the Fcregion as the second domain. In one embodiment, Fc region comoprised inthe second domain comprises a hinge region, a CH2 domain and a CH3domain.

In some embodiments, the Fc region comprised in the second domain can becan be used to facilitate expression and purification of the engineeredmicrobe-targeting molecules described herein. The N terminal Fc has beenshown to improve expression levels, protein folding and secretion of thefusion partner. In addition, the Fc has a staphylococcal Protein Abinding site, which can be used for one-step purification protein Aaffinity chromatography. See Lo K M et al. (1998) Protein Eng. 11:495-500. Further, the Protein A binding site can be used to facilitatebinding of Protein A-expressing or Protein G-expressing microbes in theabsence of calcium ions. Such binding capability can be used to developmethods for distinguishing protein A-expressing microbes (e.g., S.aureus) from non-protein A-expressing or non-protein G-expressingmicrobes (e.g., E. coli) present in a test sample, and variousembodiments of such methods will be described in detail later. Further,such Fc regions have a molecule weight above a renal threshold of about45 kDa, thus reducing the possibility of engineered microbe-targetingmolecules being removed by glomerular filtration. Additionally, the Fcregion can allow dimerization of two engineered microbe-targetingmolecules to form a multimeric complexe, such as a dimer.

In some embodiments, the second domain comprises the amino acid sequenceSEQ ID NO: 5, 6, 7 or 42.

In some embodiments, where the second domain comprises a Fc region or afragment thereof, the Fc region or a fragment thereof can comprise atleast one mutation, e.g., to modify the performance of the engineeredmicrobe-targeting molecules. For example, in some embodiments, ahalf-life of the engineered microbe-targeting molecules described hereincan be increased, e.g., by mutating an amino acid lysine (K) at theresidue 232 of SEQ ID NO. 5, 6, or 7 to alanine (A). Other mutations,e.g., located at the interface between the CH2 and CH3 domains shown inHinton et al (2004) J Biol Chem. 279:6213-6216 and Vaccaro C. et al.(2005) Nat Biotechnol. 23: 1283-1288, can be also used to increase thehalf-life of the IgG1 and thus the engineered microbe-targetingmolecules.

In some embodiments, the second domain can comprise a microbe-bindingdomain (or a fragment thereof retaining microbe binding activity) from amicrobe-binding protein. In some embodiments, the second domaincomprises a microbe-binding domain (or a fragment thereof retainingmicrobe binding activity) from microbe-binding protein that is not CRP.The terms “microbe binding domain” and “microbe surface-binding domain”are used interchangeably herein and refer to any molecule or a fragmentthereof that can specifically bind to the surface of a microbe orpathogen, e.g., any component present on a surface of a microbe orpathogen, or any matter or component/fragment that is derived,originated or secreted from a microbe or pathogen. Molecules that can beused in the microbe surface-binding domain can include, for example, butare not limited to, peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, smallmolecules, and any combinations thereof.

In some embodiments, the microbe surface-binding domain can comprise apeptidomimetic that mimics a molecule or a fragment thereof that canspecifically bind to the surface of a microbe or pathogen, or microbialmatter. For example, a microbe surface-binding domain can comprise apeptidomimetic that mimics a carbohydrate recognition domain or afragment thereof, e.g., carbohydrate recognition domain of MBL or afragment thereof.

In some embodiments, the microbe surface-binding domain can be acarbohydrate recognition domain or a fragment thereof of carbohydratebinding protein. The term “carbohydrate recognition domain” as usedherein refers to a region, at least a portion of which, can bind tocarbohydrates on a surface of microbes or pathogens. In someembodiments, the second domain can comprise at least about 50% of thefull length CRD, including at least about 60%, at least about 70%, atleast about 80%, at least about 90% or higher, capable of binding tocarbohydrates on a microbe surface. In some embodiments, 100% of thecarbohydrate recognition domain can be used to bind to microbes orpathogens. In other embodiments, the carbohydrate recognition domain cancomprise additional regions that are not capable of carbohydratebinding, but can have other characteristics or perform other functions,e.g., to provide flexibility to the carbohydrate recognition domain wheninteracting with microbes or pathogens.

Exemplary carbohydrate-binding proteins include, but are not limited to,lectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, and glucose-bindingprotein. Additional carbohydrate-binding proteins that can be includedin the microbe surface-binding domain described herein can include, butare not limited to, lectins or agglutinins that are derived from aplant, e.g., Galanthus nivalis agglutinin (GNA) from the Galanthus(snowdrop) plant, and peanut lectin. In some embodiments, pentraxinfamily members (e.g., C-reactive protein) can also be used as acarbohydrate-binding protein. Pentraxin family members can generallybind capsulated microbes. Without limitation, the carbohydrate-bindingproteins can be wild-type, recombinant or a fusion protein. Therespective carbohydrate recognition domains for suchcarbohydrate-binding proteins are known in the art, and can be modifiedfor various embodiments of the engineered microbe-targeting moleculesdescribed herein.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can be used in the engineeredmicrobe-targeting molecules. For example, recombinant mannose-bindinglectins, e.g., but not limited to, the ones disclosed in the U.S. Pat.Nos. 5,270,199; 6,846,649; U.S. Patent Application No. US 2004/0229212;and PCT Application No. WO 2011/090954, filed Jan. 19, 2011, thecontents of all of which are incorporated herein by reference, can beused in constructing the microbe-targeting molecules described herein.

In some embodiments, the CRD is from an MBL, a member of the collectinfamily of proteins. A native MBL is a multimeric structure (e.g., about650 kDa) composed of subunits, each of which contains three identicalpolypeptide chains. Each MBL polypeptide chain (containing 248 aminoacid residues in length with a signal sequence: SEQ ID NO.8) comprises aN-terminal cysteine rich region, a collagen-like region, a neck region,and a carbohydrate recognition domain (CRD). The sequence of each regionhas been identified and is well known in the art. SEQ ID NO. 8 is thefull-length amino acid sequence of MBL without a signal sequence. Insome embodiments, the signal sequence corresponds to amino acids 1-20 ofSEQ ID NO 8, i.e. SEQ ID NO: 10.

The full-length amino acid sequence of carbohydrate recognition domain(CRD) of MBL is shown in SEQ ID NO. 11. In some embodiments, thecarbohydrate recognition domain of the engineered MBL molecule cancomprise a fragment of SEQ ID NO. 11. Exemplary amino acid sequences ofsuch fragments include, but are not limited to, ND (SEQ ID NO. 12), EZN(SEQ ID NO. 13: where Z is any amino acid, e.g., P), NEGEPNNAGS (SEQ IDNO. 14) or a fragment thereof comprising EPN, GSDEDCVLL (SEQ ID NO. 15)or a fragment thereof comprising E, and LLLKNGQWNDVPCST (SEQ ID NO.16)or a fragment thereof comprising ND. Modifications to such CRDfragments, e.g., by conservative substitution, are also within the scopedescribed herein. In some embodiments, the MBL or a fragment thereofused in the the engineered microbe-targeting molecules described hereincan be a wild-type molecule or a recombinant molecule.

In some circumstances, complement or coagulation activation induced by acarbohydrate-binding protein or a fragment thereof can be undesirabledepending on various applications, e.g., in vivo administration fortreatment of sepsis. In such embodiments, the additional portion of thecarbohydrate-binding protein can exclude at least one of complement andcoagulation activation regions. By way of example, when thecarbohydrate-binding protein is mannose-binding lectin or a fragmentthereof, the mannose-binding lectin or a fragment thereof can exclude atleast one of the complement and coagulation activation regions locatedon the collagen-like region. In such embodiments, the mannose-bindinglectin or a fragment thereof can exclude at least about one amino acidresidue, including at least about two amino acid residues, at leastabout three amino acid residues, at least about four amino acidresidues, at least about five amino acid residues, at least about sixamino acid residues, at least about seven amino acid residues, at leastabout eight amino acid residues, at least about nine amino acidresidues, at least about ten amino acid residues or more, around aminoacid residue K55 or L56 of SEQ ID NO. 9. Exemplary amino sequencescomprising K55 or L56 of SEQ ID NO. 8 that can be excluded from thesecond domain of the microbe-binding molecule include, but are notlimited to, EPGQGLRGLQGPPGKLGPPGNPGPSGS (SEQ ID NO. 17), GKLG (SEQ IDNO. 18), GPPGKLGPPGN (SEQ ID NO. 19), RGLQGPPGKL (SEQ ID NO. 20),GKLGPPGNPGPSGS (SEQ ID NO. 21), GLRGLQGPPGKLGPPGNPGP (SEQ ID NO. 22), orany fragments thereof.

In some embodiments, the additional portion of the carbohydrate-bindingproteins can activate the complement system. In alternative embodiments,the additional portion of the carbohydrate-binding protein cannotactivate the complement system. In some embodiments, the additionalportion of the carbohydrate-binding protein can be selected, configured,or modified such that it does not activate the complement system.

In some embodiments, the second domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 8, 9, 11, 12, or 23-27.

In some embodiments, the second domain can comprise a neck region or afragment thereof from a lectin. By neck region of a lection is meant theportion of the lection than connects the CRD to rest of the molecule.Without wishing to be bound by theory, the neck region can provideflexibility and proper orientation to the first domain for binding to amicrobe surface. When the microbe-binding molecule disclosed hereincomprises second domain comprising a neck region and an additionalsecond domain, the neck region can be located between the first domainand the additional second domain, i.e., the neck region can act as alinker for linking the first domain and the additional second domain. Insome embodiments, the second domain can comprise one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, or more)additional amino acids on the N- or C-terminus of the neck region. Insome embodiments, the neck region comprises the amino acid sequence

(SEQ ID NO: 28) PDGDSSLAASERKALQTEMARIKKWLTFSLGKQ, (SEQ ID NO: 29)APDGDSSLAASERKALQTEMARIKKWLTFSLGKQ, (SEQ ID NO: 30)PDGDSSLAASERKALQTEMARIKKWLTFSLG, or (SEQ ID NO: 31)APDGDSSLAASERKALQTEMARIKKWLTFSLG.

In some embodiments, the second domain can comprise a detectable label.As used herein, the term “detectable label” refers to a compositioncapable of producing a detectable signal indicative of the presence of atarget. Detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable labels include fluorescentmolecules, radioisotopes, nucleotide chromophores, enzymes, substrates,chemiluminescent moieties, bioluminescent moieties, and the like. Assuch, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means needed for the methods and devices described herein.

In some embodiments, the detectable label can be an imaging agent orcontrast agent. As used herein, the term “imaging agent” refers to anelement or functional group in a molecule that allows for the detection,imaging, and/or monitoring of the presence and/or progression of acondition(s), pathological disorder(s), and/or disease(s). The imagingagent can be an echogenic substance (either liquid or gas), non-metallicisotope, an optical reporter, a boron neutron absorber, a paramagneticmetal ion, a ferromagnetic metal, a gamma-emitting radioisotope, apositron-emitting radioisotope, or an x-ray absorber. As used herein theterm “contrast agent” refers to any molecule that changes the opticalproperties of tissue or organ containing the molecule. Opticalproperties that can be changed include, but are not limited to,absorbance, reflectance, fluorescence, birefringence, optical scatteringand the like. In some embodiments, the detectable labels also encompassany imaging agent (e.g., but not limited to, a bubble, a liposome, asphere, a contrast agent, or any detectable label described herein) thatcan facilitate imaging or visualization of a tissue or an organ in asubject, e.g., for diagnosis of an infection.

Suitable optical reporters include, but are not limited to, fluorescentreporters and chemiluminescent groups. A wide variety of fluorescentreporter dyes are known in the art. Typically, the fluorophore is anaromatic or heteroaromatic compound and can be a pyrene, anthracene,naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole,benzothiazole, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluorescein, rhodamine or other like compound.

Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); ALEXA FLUOR 350™(7-Amino-4-methyl-6-sulfocoumarin-3-acetic acid); ALEXA FLUOR 430™;ALEXA FLUOR 488™; ALEXA FLUOR 532™; ALEXA FLUOR 546™; ALEXA FLUOR 568™;ALEXA FLUOR 594™; ALEXA FLUOR 633™; ALEXA FLUOR 647™; ALEXA FLUOR 660™;ALEXA FLUOR 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin(APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; AminoactinomycinD; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS;ASTRAZON Brilliant Red 4G (basic red 14); ASTRAZON Orange R(2-[2-(1-Methyl-2-phenyl-1H-indol-3-yl)ethenyl]-1,3,3-trimethyl-3H-indoliumchloride);ASTRAZON Red 6B (basic violet 7); ASTRAZON Yellow 7 GLL; Atabrine;ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine;BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); BG-647;Bimane; Bisbenzamide; BLANCOPHOR FFG (7-diethylamino-4-methylcoumarin);BLANCOPHOR SV; BOBO™-1; BOBO™-3; BODIPY 492/515; BODIPY 493/503(4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene);BODIPY 500/510; BODIPY 505/515(4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene); BODIPY530/550; BODIPY 542/563; BODIPY 558/568; BODIPY 564/570; BODIPY 576/589;BODIPY 581/591; BODIPY 630/650-X; BODIPY 650/665-X; BODIPY 665/676;BODIPY Fl; BODIPY FL ATP; BODIPY Fl-Ceramide; BODIPY R6G SE; BODIPY TMR;BODIPY TMR-X conjugate; BODIPY TMR-X, SE; BODIPY TR; BODIPY TR ATP;BODIPY TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF;Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX);Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP—Cyan FluorescentProtein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA;Coelenterazine ; Coelenterazine cp; Coelenterazine f; Coelenterazinefcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip;Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™;Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMPFluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO(DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride;Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645;Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wildtype, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP);GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258;Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin EBG; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 ; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200 ; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodamineIsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary detectable labels include luminescent and bioluminescentmarkers (e.g., biotin, luciferase (e.g., bacterial, firefly, clickbeetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H,125I, 35S, 14C, or 32P), enzymes (e.g., galactosidases, glucorinidases,phosphatases (e.g., alkaline phosphatase), peroxidases (e.g.,horseradish peroxidase), and cholinesterases), and calorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149, and 4,366,241, each of which is incorporatedherein by reference.

Suitable echogenic gases include, but are not limited to, a sulfurhexafluoride or perfluorocarbon gas, such as perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluropentane, or perfluorohexane. Suitablenon-metallic isotopes include, but are not limited to, ¹¹C, ¹⁴C, ¹³N,¹⁸F, ¹²³I, ¹²⁴I, and ¹²⁵I. Suitable radioisotopes include, but are notlimited to, ⁹⁹mTc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, Ga, ⁶⁸Ga, and ¹⁵³Gd.Suitable paramagnetic metal ions include, but are not limited to,Gd(III), Dy(III), Fe(III), and Mn(II). Suitable X-ray absorbers include,but are not limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au,Yb, Dy, Cu, Rh, Ag, and Ir.

In some embodiments, the radionuclide is bound to a chelating agent orchelating agent-linker attached to the microbe-targeting molecule.Suitable radionuclides for direct conjugation include, withoutlimitation, ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, and mixtures thereof. Suitableradionuclides for use with a chelating agent include, withoutlimitation, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In,¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, andmixtures thereof. Suitable chelating agents include, but are not limitedto, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phosphonate analogs,and mixtures thereof. One of skill in the art will be familiar withmethods for attaching radionuclides, chelating agents, and chelatingagent-linkers to molecules such as the microbe-targeting molecules andcarrier scaffolds disclosed herein.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label. Exemplary methods for in vivo detectionor imaging of detectable labels include, but are not limited to,radiography, magnetic resonance imaging (MRI), Positron emissiontomography (PET), Single-photon emission computed tomography (SPECT, orless commonly, SPET), Scintigraphy, ultrasound, CAT scan, photoacousticimaging, thermography, linear tomography, poly tomography, zonography,orthopantomography (OPT or OPG), and computed Tomography (CT) orComputed Axial Tomography (CAT scan).

In some embodiments, the detectable label can include an enzyme.Exemplary enzymes for use as detectable labels include, but are notlimited to, horseradish peroxidase (HRP), alkaline phosphastase (AP), orany combinations thereof.

In some embodiments, the detectable can include a microbial enzymesubstrate conjugated to a detectable agent. For example, the detectableagent can be any moiety that, when cleaved from a microbial enzymesubstrate by the enzyme possessed or secreted by the microbe, forms adetectable moiety but that is not detectable in its conjugated state.The microbial enzyme substrate is a substrate specific for one or moretypes of microbes to be detected, and it can be selected depending uponwhat enzymes the microbe possesses or secretes. See, e.g., InternationalPatent Application: WO 2011/103144 for the use of such detectable labelin detection of microbes, the content of which is incorporated herein byreference.

In some embodiments, the detectable label is a fluorophore or a quantumdot. Without wishing to be bound by a theory, using a fluorescentreagent can reduce signal-to-noise in the imaging/readout, thusmaintaining sensitivity. Accordingly, in some embodiments, prior todetection, the microbes isolated from or remained bound on themicrobe-targeting substrate can be stained with at least one stain,e.g., at least one fluorescent staining reagent comprising amicrobe-binding molecule, wherein the microbe-binding molecule comprisesa fluorophore or a quantum dot. Examples of fluorescent stains include,but are not limited to, any microbe-targeting element (e.g.,microbe-specific antibodies or any microbe-binding proteins or peptidesor oligonucleotides) typically conjugated with a fluorophore or quantumdot, and any fluorescent stains used for detection as described herein.In some embodiments, the detectable label is a gold particle.

In some embodiments, the detectable label can be configured to include a“smart label”, which is undetectable when conjugated to themicrobe-binding molecules, but produces a color change when releasedfrom the engineered molecules in the presence of a microbe enzyme. Thus,when a microbe binds to the engineered microbe-binding molecules, themicrobe releases enzymes that release the detectable label from theengineered molecules. An observation of a color change indicatespresence of the microbe in the sample.

In some embodiments, the detectable label can be a chromogenic orfluorogenic microbe enzyme substrate so that when a microbe binds to theengineered microbe-targeting molecule, the enzyme that the microbereleases can interact with the detectable label to induce a colorchange. Examples of such microbe enzyme substrate can include, but arenot limited to, indoxyl butyrate, indoxyl glucoside, esculin, magnetaglucoside, red-β-glucuronide, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-glu-copyranoside, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-cetamindo-2-deoxyglucopyranoside, and any other art-recognizedmicrobe enzyme substrates. Such embodiments can act as an indicator forthe presence of a microbe or pathogen.

In some embodiments, the second domain can comprise a functional groupfor conjugating the first domain to another molecule, a composition, aphysical substrate, and the like. For example, the second domain cancomprise a functional group for covalently linking the first domain withanother molecule molecule, a composition, a physical substrate, or thelike. Some exemplary functional groups for conjugation include, but arenot limited to, an amino group, a N-substituted amino group, a carboxylgroup, a carbonyl group, an acid anhydride group, an aldehyde group, ahydroxyl group, an epoxy group, a thiol, a disulfide group, an alkenylgroup, a hydrazine group, a hydrazide group, a semicarbazide group, athiosemicarbazide group, one partner of a binding pair, an amide group,an aryl group, an ester group, an ether group, a glycidyl group, a halogroup, a hydride group, an isocyanate group, an urea group, an urethanegroup, and any combinations thereof.

In some embodiments, the microbe-binding molecule disclosed herein canbe immobilized on a carrier scaffold for a variety of applications orpurposes. For example, when the affinity of a single microbesurface-binding domain for a target molecule is relatively low, and suchbinding is generally driven by avidity and multivalency, multivalency ofthe engineered microbe-targeting molecules disclosed herein can beeffectively increased by attachment of a plurality of the engineeredmicrobe-targeting molecules to a carrier scaffold, such as a solidsubstrate at a high density, which can be varied to provide optimalfunctionality. Alternatively, the engineered microbe-targeting moleculescan be immobilized on a carrier scaffold for easy handling during usage,e.g., for isolation, observation or microscopic imaging.

The attachment of the engineered microbe-binding molecule disclosedherein to a surface of the carrier scaffold can be performed withmultiple approaches, for example, by direct cross-linking the engineeredmicrobe-binding molecule to the carrier scaffold surface; cross-linkingthe engineered microbe-binding molecule to the carrier scaffold surfacevia a nucleic acid matrix (e.g., DNA matrix or DNA/oligonucleotideorigami structures) for orientation and concentration to increasedetection sensitivity; cross-linking the microbe-binding molecule to thecarrier scaffold surface via a dendrimer-like structure (e.g.,PEG/Chitin-structure) to increase detection sensitivity; attractingmicrobe-binding molecule coated magnetic microbeads to the carrierscaffold surface with a focused magnetic field gradient applied to thescarrier scaffold surface, attaching an engineered microbe-bindingmolecule to a carrier scaffold via biotin-avidin or biotin-avidin-likeinteraction, or any other art-recognized methods.

Without limitations, any conjugation chemistry known in the art forconjugating two molecules or different parts of a composition togethercan be used for conjugating at least one engineered microbe-targetingmolecule to a carrier scaffold. Exemplary coupling molecules and/orfunctional groups for conjugating at least one engineeredmicrobe-targeting molecule to a substrate include, but are not limitedto, a polyethylene glycol (PEG, NH₂—PEG_(x)-COOH which can have a PEGspacer arm of various lengths X, where 1<X<100, e.g., PEG-2K, PEG-5K,PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K, and the like), maleimideconjugation agent, PASylation, HESylation, Bis(sulfosuccinimidyl)suberate conjugation agent, DNA conjugation agent, peptide conjugationagent, silane conjugation agent, polysaccharide conjugation agent,hydrolyzable conjugation agent, and any combinations thereof.

For engineered microbe-targeting molecules to be immobilized on orconjugated to a carrier scaffold, the microbe-targeting moleculesdescribed herein can further comprise at least one (e.g., one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty or more) second domain, e.g., adapted for orienting the firstdomain away from the carrier scaffold surface. In some embodiments, thecarrier scaffold surface can be functionalized with a coupling moleculeto facilitate the conjugation of engineered microbe-targeting moleculeto the solid surface.

Accordingly, in some embodiments, the second domain can be selected orconfigured to provide one or more functional groups for conjugating themicrobe-binding domain with a carrier scaffold or a deteactable label. Adomain adapted for conjugating the microbe-binding molecule to a carrierscaffold is also referred to as a “conjugation domain” herein. As usedherein, the term “conjugation domain” refers to any molecule or portionthereof that facilitates the conjugation of the engineered moleculesdescribed herein to a carrier scaffold.

In some embodiments, length of the conjugation domain can vary from 1amino acid residue to about 10 amino acid residues, or about 2 aminoacid residues to about 5 amino acid residues. Determination of anappropriate amino acid sequence of the oconjugatio domain for bindingwith different carrier scaffolds is well within one of skill in the art.For example, according to one or more embodiments, the conjugationdomain can comprise an amino acid sequence of AKT (SEQ ID NO: 32), whichprovides a single biotinylation site for subsequent binding tostreptavidin. Preferably the AKT is at the terminus or near the terminus(e.g., within less than 10 amino acids from the terminus) of themicrobe-binding molecule. In some embodiments, the conjugation domaincomprises a functional group for conjugating or linking themicrobe-binding molecule to the carrier scaffold. Some exemplaryfunctional groups for conjugation include, but are not limited to, anamino group, a N-substituted amino group, a carboxyl group, a carbonylgroup, an acid anhydride group, an aldehyde group, a hydroxyl group, anepoxy group, a thiol, a disulfide group, an alkenyl group, a hydrazinegroup, a hydrazide group, a semicarbazide group, a thiosemicarbazidegroup, one partner of a binding pair, an amide group, an aryl group, anester group, an ether group, a glycidyl group, a halo group, a hydridegroup, an isocyanate group, an urea group, an urethane group, and anycombinations thereof.

Activation agents can be used to activate the components to beconjugated together. Without limitations, any process and/or reagentknown in the art for conjugation activation can be used. Exemplaryactivation methods or reagents include, but are not limited to,1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC orEDAC), hydroxybenzotriazole (HOBT), N-Hydroxysuccinimide (NHS),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (HATU), silanization, surfaceactivation through plasma treatment, and the like.

In some embodiments, the conjugation domain can comprise at least oneamino group that can be non-convalently or covalently coupled withfunctional groups on the carrier scaffold. For example, the primaryamines of the amino acid residues (e.g., lysine or cysteine residues)can be used to conjugate the microbe-binding molecule with the carrierscaffold. In some embodiments, the amino group at the N-terminus of themicrobe-binding molecule can be used for conjugating the microbe-bindingmolecule with the carrier scaffold.

Without limitations, the engineered microbe-targeting molecule can beconjugated to the carrier-scaffold through covalent or non-covalentinteractions or any combination of covalent and non-covalentinteractions. Further, conjugation can be accomplished any of methodknown to those of skill in the art. For example, covalent immobilizationcan be accomplished through, for example, silane coupling. See, e.g.,Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol.134 (1976). The covalent interaction between the engineeredmicrobe-targeting molecule and/or coupling molecule and the surface canalso be mediated by other art-recognized chemical reactions, such as NHSreaction or a conjugation agent. The non-covalent interaction betweenthe engineered microbe-targeting molecule and/or coupling molecule andthe surface can be formed based on ionic interactions, van der Waalsinteractions, dipole-dipole interactions, hydrogen bonds, electrostaticinteractions, and/or shape recognition interactions.

Without limitations, conjugation can include either a stable or a labile(e.g. cleavable) bond or conjugation agent. Exemplary conjugationsinclude, but are not limited to, covalent bond, amide bond, additions tocarbon-carbon multiple bonds, azide alkyne Huisgen cycloaddition,Diels-Alder reaction, disulfide linkage, ester bond, Michael additions,silane bond, urethane, nucleophilic ring opening reactions: epoxides,non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolarcycloaddition, temperature sensitive, radiation (IR, near-IR, UV)sensitive bond or conjugation agent, pH-sensitive bond or conjugationagent, non-covalent bonds (e.g., ionic charge complex formation,hydrogen bonding, pi-pi interactions, hist guest interactions, such ascyclodextrin/adamantly host guest interaction) and the like.

In some embodiments, the microbe-targeting molecule can be conjugated tothe carrier-scaffold with a linker. In some embodiments, the themicrobe-targeting molecule can be conjugated to the carrier-scaffoldwith a linking group selected from the group consisting of a directbond, an atom such as oxygen or sulfur, C(O), C(O)O, OC(O)O, C(O)NH,NHC(O)O, NH, SS, SO, SO₂, SO₃, and SO₂NH.

In some embodiments, the engineered microbe-targeting molecule can beconjugated to the carrier scaffold by a coupling molecule pair. Theterms “coupling molecule pair” and “coupling pair” as usedinterchangeably herein refer to the first and second molecules thatspecifically bind to each other. One member of the binding pair isconjugated with the carrier scaffold while the second member isconjugated with the microbe-targeting molecule. As used herein, thephrase “first and second molecules that specifically bind to each other”refers to binding of the first member of the coupling pair to the secondmember of the coupling pair with greater affinity and specificity thanto other molecules. Exemplary coupling molecule pairs include, withoutlimitations, any haptenic or antigenic compound in combination with acorresponding antibody or binding portion or fragment thereof (e.g.,digoxigenin and anti-digoxigenin; mouse immunoglobulin and goatantimouse immunoglobulin) and nonimmunological binding pairs (e.g.,biotin-avidin, biotin-streptavidin), hormone (e.g., thyroxine andcortisol-hormone binding protein), receptor-receptor agonist,receptor-receptor antagonist (e.g., acetylcholine receptor-acetylcholineor an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzymecofactor, enzyme-enzyme inhibitor, and complementary oligonucleotidepairs capable of forming nucleic acid duplexes). The coupling moleculepair can also include a first molecule that is negatively charged and asecond molecule that is positively charged.

One example of using coupling pair conjugation is the biotin-avidin orbiotin-streptavidin conjugation. In this approach, one of the members ofmolecules to be conjugated together (e.g., the engineeredmicrobe-targeting molecule or the carrier scaffold) is biotinylated andthe other is conjugated with avidin or streptavidin. Many commercialkits are available for biotinylating molecules, such as proteins. Forexample, an aminooxy-biotin (AOB) can be used to covalently attachbiotin to a molecule with an aldehyde or ketone group. In someembodiments, AOB is attached to the engineered microbe-targetingmolecule. Further, as described elsewhere herein, an AKT sequence on theN-terminal of the engineered microbe-targeting molecule can allow theengineered microbe-targeting molecule to be biotinylated at a singlesite and further conjugated to the streptavidin-coated solid surface.Moreover, the microbe-binding molecule can be coupled to a biotinacceptor peptide, for example, the AviTag or Acceptor Peptide (referredto as AP; Chen et al., 2 Nat. Methods 99 (2005)). The Acceptor Peptidesequence allows site-specific biotinylation by the E. coli enzyme biotinligase (BirA; Id.). Thus, in some embodiments, the conjugation domaincomprises an amino acid sequence of a biotin acceptor peptide.

Another non-limiting example of using conjugation with a couplingmolecule pair is the biotin-sandwich method. See, e.g., Davis et al.,103 PNAS 8155 (2006). In this approach, the two molecules to beconjugated together are biotinylated and then conjugated together usingtetravalent streptavidin. Another example for conjugation would be touse PLP-mediated bioconjugation. See, e.g., Witus et al., 132 JACS 16812(2010). Still another example of using coupling pair conjugation isdouble-stranded nucleic acid conjugation.

In this approach, one of the members of molecules to be conjugatedtogether is conjugated with a first strand of the double-strandednucleic acid and the other is conjugated with the second strand of thedouble-stranded nucleic acid. Nucleic acids can include, withoutlimitation, defined sequence segments and sequences comprisingnucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs,modified nucleotides and nucleotides comprising backbone modifications,branchpoints and nonnucleotide residues, groups or bridges.

The carrier scaffold can also be functionalized to include a functionalgroup for conjugating with the microbe-binding molecule. In someembodiments, the carrier scaffold can be functionalized to include acoupling molecule, or a functional fragment thereof, that is capable ofselectively binding with an engineered microbe-targeting moleculedescribed herein, As used herein, the term “coupling molecule” refers toany molecule or any functional group that is capable of selectivelybinding with an engineered microbe surface-binding domain describedherein. Representative examples of coupling molecules include, but arenot limited to, antibodies, antigens, lectins, proteins, peptides,nucleic acids (DNA, RNA, PNA and nucleic acids that are mixtures thereofor that include nucleotide derivatives or analogs); receptor molecules,such as the insulin receptor; ligands for receptors (e.g., insulin forthe insulin receptor); and biological, chemical or other molecules thathave affinity for another molecule.

In some embodiments, the coupling molecule is an aptamer. As usedherein, the term “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules by a mechanism otherthan Watson-Crick base pairing or triplex formation. Aptamers caninclude, without limitation, defined sequence segments and sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges. Methods for selecting aptamers for binding to a molecule arewidely known in the art and easily accessible to one of ordinary skillin the art. The aptamers can be of any length, e.g., from about 1nucleotide to about 100 nucleotides, from about 5 nucleotides to about50 nucleotides, or from about 10 nucleotides to about 25 nucleotides.

In some embodiments, the second domain comprises a therapeutic agent.For example, the second domain can comprise an anti-microbial agent.Therapeutic agents are described herein below. Any method available tothe skilled artisan for conjugating a therapeutic agent to a peptide canbe used for conjugating the therapeutic agent to the first domain. Forexample, functional groups or methods used for conjugating themicrobe-targeting molecule to a carrier scaffold can also be used forconjugating the microbe-targeting molecule to a therapeutic agent. Thiscan be beneficial for delivering or concentrating a therapeutic agent(e.g., an anti-microbial agent) at a nidus of infection.

The first and second domains of the microbe-targeting molecule arelinked together by a linker. Further, the microbe-targeting molecule canbe conjugated to a carrier scaffold via linker. Accordingly, as used inthis disclosure, the term “linker” means a moiety that connects twoparts of a compound or molecule. Linkers typically comprise a directbond or an atom such as oxygen or sulfur, a unit such as NR¹, C(O),C(O)O, OC(O)O, C(O)NH, NHC(O)O, NH, SS, SO, SO₂, SO₃, and SO₂NH, or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, NH,C(O)N(R¹)₂, C(O), cleavable linking group, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R¹ is hydrogen, acyl, aliphatic orsubstituted aliphatic. In some embodiments, the linker can be anon-covalent association (e.g., by non-covalent interactins) of the twoparts of a molecule being conjugated together. Some exemplarynon-covalent on ionic interactions, van der Waals interactions,dipole-dipole interactions, hydrogen bonds, electrostatic interactions,and/or shape recognition interactions.

In some embodiments, the linker can comprise at least one cleavablelinking group. A cleavable linking group is one which is sufficientlystable under one set of conditions, but which is cleaved under adifferent set of conditions to release the two parts the linker isholding together. In some embodiments, the cleavable linking group iscleaved at least 10 times or more, e.g., at least 100 times faster undera first reference condition (which can, e.g., be selected to mimic orrepresent a microbe-infected condition, such as a microbe-infectedtissue or body fluid, or a microbial biofilm occurring in anenvironment) than under a second reference condition (which can, e.g.,be selected to mimic or represent non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).

Cleavable linking groups are susceptible to cleavage agents, e.g.,hydrolysis, pH, redox potential or the presence of degradativemolecules. Generally, cleavage agents are more prevalent or found athigher levels or activities at a site of interest (e.g. a microbialinfection) than in non-infected area. Examples of such degradativeagents include: redox agents which are selected for particularsubstrates or which have no substrate specificity, including, e.g.,oxidative or reductive enzymes or reductive agents such as mercaptans,present in cells, that can degrade a redox cleavable linking group byreduction; esterases; amidases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific)and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell, organ, or tissue to be targeted. Insome embodiments, cleavable linking group is cleaved at least 1.25, 1.5,1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster under a firstreference condition (or under in vitro conditions selected to mimic amicrobe-infected condition, such as a microbe-infected tissue or bodyfluid, or a microbial biofilm occurring in an environment or on aworking surface) than under a second reference condition (or under invitro conditions selected to mimic non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).In some embodiments, the cleavable linking group is cleaved by less than90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in thenon-infected conditions, e.g., found in the non-infected blood or serum,or in an non-infected environment, as compared to a microbe-infectedcondition, such as a microbe-infected tissue or body fluid, or amicrobial biofilm occurring in an environment or on a working surface.

Exemplary cleavable linking groups include, but are not limited to,hydrolyzable linkers, redox cleavable linking groups (e.g., —S—S— and—C(R)₂—S—S—, wherein R is H or C₁-C₆ alkyl and at least one R is C₁-C₆alkyl such as CH₃ or CH₂CH₃); phosphate-based cleavable linking groups(e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—,—O—P(O)(OR)—S—, —S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—,—O—P(O)(R)—O—, —O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—,—S—P(O)(R)—S—, —O—P(S)(R)—S—, .—O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, whereinR is optionally substituted linear or branched C₁-C₁₀ alkyl); acidcleavable linking groups (e.g., hydrazones, esters, and esters of aminoacids, —C═NN— and —OC(O)—); ester-based cleavable linking groups (e.g.,—C(O)O—); peptide-based cleavable linking groups, (e.g., linking groupsthat are cleaved by enzymes such as peptidases and proteases in cells,e.g., —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the Rgroups of the two adjacent amino acids). A peptide based cleavablelinking group comprises two or more amino acids. In some embodiments,the peptide-based cleavage linkage comprises the amino acid sequencethat is the substrate for a peptidase or a protease. In someembodiments, an acid cleavable linking group is cleavable in an acidicenvironment with a pH of about 6.5 or lower (e.g., about 6.5, 6.0, 5.5,5.0, or lower), or by agents such as enzymes that can act as a generalacid.

Without limitations, the linker can be selected to provide a desiredfunction or property to the microbe-binding molecules disclosed herein.For example, the linker can be selected or configured according to aspecific need or use of the microbe-binding molecule. By way of exampleonly, in some embodiments, linker can be selected or configured to havea sufficient length and flexibility such that it can allow for the firstdomain to orient in a desired orientation with respect to a microbe. Insome embodiments, the linker can be selected or configured to allowmultimerization of at least two engineered microbe-targeting molecules(e.g., to from a di-, tri-, tetra-, penta-, hexa- or higher multimericcomplex) while retaining biological activity (e.g., microbe-bindingactivity). In some embodiments, the linker can be selected or configuredto interact with the second domain to allow multimerization of at leasttwo engineered microbe-targeting molecules (e.g., to from a di-, tri-,tetra-, penta-, hexa- or higher multimeric complex) while retainingmicrobe-binding activity.

In some embodiments, the linker can be selected or configured tofacilitate expression and purification of the engineeredmicrobe-targeting molecule described herein. In some embodiments, thelinker can be selected or configured to provide a recognition site for aprotease or a nuclease. In addition, the linker can be non-reactive withthe functional components of the engineered molecule described herein.For example, minimal hydrophobic or charged character to react with thefirst domain or second domain. In some embodiments, the linker can bepart of the first domain or second domain.

In some embodiments, the linker can be a peptide or a nucleic acid. Insome embodiments, the peptide linker can vary from about 1 to about 1000amino acids long, from about 10 to about 500 amino acids long, fromabout 30 to about 300 amino acids long, or from about 50 to about 150amino acids long. In some embodiments, the peptidyl linker is from about1 amino acid to about 20 amino acids long. In some embodiments, thenucleic acid linker can vary from about 1 to about 1000 nucleotideslong, from about 10 to about 500 nucleotides long, from about 30 toabout 300 nucleotides, or from about 50 to about 150 nucleotides. Longeror shorter linker sequences can be also used for the engineeredmicrobe-targeting molecules described herein.

The peptidyl linker can be configured to have a sequence comprising atleast one of the amino acids selected from the group consisting ofglycine (Gly), serine (Ser), asparagine (Asn), threonine (Thr),methionine (Met) or alanine (Ala). Such amino acids are generally usedto provide flexibility of a linker. However, in some embodiments, otheruncharged polar amino acids (e.g., Gln, Cys or Tyr), nonpolar aminoacids (e.g., Val, Leu, Ile, Pro, Phe, and Trp). In alternativeembodiments, polar amino acids can be added to modulate the flexibilityof a linker. One of skill in the art can control flexibility of a linkerby varying the types and numbers of residues in the linker. See, e.g.,Perham, 30 Biochem. 8501 (1991); Wriggers et al., 80 Biopolymers 736(2005).

In some embodiments, the peptidyl linker can comprise form 1 to about 25amino acids, i.e., one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three,twenty-four, or twenty-five amino acids. In some embodiments, thepeptidyl linker linking the first and second domain comprises the aminoacid sequence HHHHHH (SEQ ID NO: 39).

In some embodiments, when the second domain comprises a Fc region, thelinker linking the first and the second domain is not a bond or apeptide.

In some embodiments, the linker is a bond.

In some embodiments, the linker conjugating a microbe-targeting moleculeto a carrier scaffold is a polyethylene glycol. Exemplary PEGS for useas linkers include, but are not limited to, PEG-2K, PEG-5K, PEG-10K,PEG-12K, PEG-15K, PEG-20K, PEG-40K, and the like.

In some embodiments, the linker can be albumin, transferrin or afragment thereof. Without limitations, such linkers can be used toextend the plasma half-life of the engineered microbe-targetingmolecules. Thus, engineered microbe-targeting molecules can be usefulfor in vivo administration. See Schmidt S R (2009) Curr Opin Drug DiscovDevel. 12: 284. In some embodiments, the linker can be a physicalsubstrate, e.g., microparticles or magnetic microbes.

The linker between the first domain and the second domain can providesufficient distance between the first and the second domain to allow thefirst domain to interact with the microbes. Accordingly, the distancebetween the first domain and the second domain can range from about 50angstroms to about 5000 angstroms, from about 100 angstroms to about2500 angstroms, or from about 200 angstroms to about 1000 angstroms.

The linkers can be of any shape. For example, the linker can be linear,folded, branched. In some embodiments, the linker can adopt the shape ofa carrier scaffold. In some embodiments, the linkers can be linear. Insome embodiments, the linkers can be folded. In some embodiments, thelinkers can be branched. For branched linkers, each branch of a microbesurface-binding domain can comprise at least one microbe surface-bindingdomain. In other embodiments, the linker adopts the shape of thephysical substrate.

In some embodiments, the linker can further comprise a detectable label.In some embodiments, the detectable label can be a chromogenic orfluorogenic microbe enzyme substrate so that when a microbe binds to theengineered microbe-targeting molecule, the enzyme that the microbereleases can interact with the detectable label to induce a colorchange. Examples of such microbe enzyme substrate can include, but arenot limited to, indoxyl butyrate, indoxyl glucoside, esculin, magnetaglucoside, red-β-glucuronide, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-glucopyranoside, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-cetamindo-2-deoxyglucopyranoside, and any other art-recognizedmicrobe enzyme substrates. Such embodiments can act as an indicator forthe presence of a microbe or pathogen.

The first and second domains can be arranged in any desired orientationin the engineered microbe-targeting molecule. For example, N-terminus ofthe first domain can be linked to the C-terminus of the second domain orC-terminus of the first domain can be linked to the N-terminus of thesecond domain. It is understood that linking between the first andsecond domain is via the linker.

Further, as disclosed herein, an engineered microbe-targeting moleculecan comprise at least one microbe surface-binding domain, including atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten or moremicrobe surface-binding domains. When more than two first or seconddomains are present, such domains can all be the same, all different, orsome same and some different.

In some embodiments, the engineered microbe-targeting molecule disclosedherein comprises two or more first domains and one second domain. Insuch molecules, one first domain can be linked to the second domain andthe other first domains can be linked to the first domain linked to thesecond domain. Alternatively, two first domains can be linked to thesecond domain and other first domains can be linked to one or both ofthe two first domains linked to the second domain.

In some embodiments, the engineered microbe-targeting molecule disclosedherein comprises two or more second domains and one first domain. Insuch molecules, one second domain can be linked to the first domain andthe other second domains can be linked to the second domain linked tothe first domain. Alternatively, two second domains can be linked to thefirst domain and other second domains can be linked to one or both ofthe two second domains linked to the first domain.

In some embodiments, the engineered mincrobe-targeting molecule is inthe form of a multimeric complex comprising at least two (e.g., two,three, four, five, six, seven, eight, nine, ten, or more) engineeredmicrobe-targeting molecules. Accordingly, the multimeric complex can bea di-, tri-, tetra-, penta-, hexa- or higher multimeric complex. In oneembodiment, the engineered mincrobe-targeting molecule is in the form ofpentameric complex. Without limitations, the multimeric complex can beformed by interactions between a second domain or linker of a firstmolecule with a second domain or a linker of the second molecule. Suchinteractions can comprise covalent linking or non-covalent linking. Themicrobe-targeting molecules in the multimeric complex can all be thesame, all different, or some same and some different.

In some embodiments, the second domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5-9, 11, 12, 23-32,39, and any combinations thereof.

General methods of preparing any embodiments of the engineeredmicrobe-targeting molecules are known in the art (Ashkenazi, A. and S.M. Chamow (1997), “Immunoadhesins as research tools and therapeuticagents,” Curr. Opin. Immunol. 9(2): 195-200, Chamow, S. M. and A.Ashkenazi (1996). “Immunoadhesins: principles and applications,” TrendsBiotechnol. 14(2):52-60). In one example, an engineeredmicrobe-targeting molecule can be made by cloning into an expressionvector such as Fc-X vector as discussed in Lo et al. (1998) 11:495.

While the exemplary sequences provided herein are derived from a humanspecies, amino acid sequences for same or functionally equivalentdomains from other species such as mice, rats, porcine, bovine, feline,and canine are known in the art and within the scope described herein.Further, a skill artisan can readily modify the identified sequences tomodulate their orientation or binding performance, e.g., by theoreticalmodeling or in vitro binding experiments. In addition, based on thecrystal structure of the native sequences, peptidomimetics that caneffectively mimic at least a fragment of a given domain can be also usedas a first or second domain of the engineered microbe-targeting moleculedescribed herein. One of skill in the art can readily determine suchpeptidomimetic structure without undue experimentations, using anymethods known in the art and the known crystal structure.

In another strategy of directed evolution, the protein of interest issubjected to random mutagenesis and the resulting proteins are screenedfor desired qualities. This is a particularly useful technology foraffinity maturation of phage display antibodies, where the antibodycomplementary determining regions (CDRs) are mutated by saturationmutagenesis and successful variants of the six CDRs are shuffledtogether to form the highest affinity antibodies.

The directed evolution paradigm can be applied to any domain describedherein to select variants with a desired property, such as pecificbinding to, e.g., but not limited to, yeast, gram-positive bacteria,gram-negative, coagulase negative, and aerobic bacteria. For this towork, however, the pattern and nature of the target sugars or relatedsurface features on these target microorganisms can differ between theclasses or species.

Derivatives with a particular specificity can be isolated, e.g., by thefollowing approach, which is a standard phage display strategy: First,express a set of variants from a phagemid vector; then bind this libraryto a target of interest and perform one or two rounds of selection; andthen perform a round of negative selection against a related target,taking those phagemids that fail to bind. These cycles of positive andnegative selection are then repeated until a population of phages thatgenerally bind to the target and do not bind to the non-target isgenerated. This method can be applied to any pair of microbial strainsagainst which differential binding is desired, such as bacteria that areresistant and sensitive to a given antibiotic. This positive/negativeenrichment strategy can also be used with an antibody-phage displaylibrary, which is an even more standard way to isolate such specificbinders.

The inventors have also discovered inter alia that expression ofpeptides containing a portion of a c-reactive protein can be increasedby modifying the nucleic acid encoding the c-reactive protein.Accordingly, provided herein are nucleic acid sequences for theexpression of CRP containing peptides that are expressed in an higheramount relative to expression from a wild-type nucleic acid encodingCRP, such as from SEQ ID NO: 52. In some embodiments, the CRP isexpressed from a nucleic acid sequence comprising SEQ ID NO: 45, 46, 47,48, 49, 50, 51 or any combinations thereof.

In some embodiments, the nucleic acid sequence described herein isexpressed in a recombinant expression vector or plasmid. As used herein,the term “vector” refers to a polynucleotide sequence suitable fortransferring transgenes into a host cell. The term “vector” includesplasmids, mini-chromosomes, phage, naked DNA and the like. See, forexample, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828;5,888,783 and, 5,919,670, and, Sambrook et al, Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type ofvector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments are ligated. Another type ofvector is a viral vector, wherein additional DNA segments are ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Moreover, certain vectors are capable of directingthe expression of genes to which they are operatively linked. Suchvectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” is used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

A cloning vector is one which is able to replicate autonomously orintegrated in the genome in a host cell, and which is furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence can be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence can occur many times as the plasmidincreases in copy number within the host cell such as a host bacteriumor just a single time per host before the host reproduces by mitosis. Inthe case of phage, replication can occur actively during a lytic phaseor passively during a lysogenic phase.

An expression vector is one into which a desired DNA sequence can beinserted by restriction and ligation such that it is operably joined toregulatory sequences and can be expressed as an RNA transcript. Vectorscan further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase, luciferase or alkaline phosphatase),and genes which visibly affect the phenotype of transformed ortransfected cells, hosts, colonies or plaques (e.g., green fluorescentprotein). In certain embodiments, the vectors used herein are capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript can be translated into thedesired protein or polypeptide.

When the nucleic acid molecule that encodes any of the nucleic acidsdescribed herein is expressed in a cell, a variety of transcriptioncontrol sequences (e.g., promoter/enhancer sequences) can be used todirect its expression. The promoter can be a native promoter, i.e., thepromoter of the gene in its endogenous context, which provides normalregulation of expression of the gene. In some embodiments the promotercan be constitutive, i.e., the promoter is unregulated allowing forcontinual transcription of its associated gene. A variety of conditionalpromoters also can be used, such as promoters controlled by the presenceor absence of a molecule.

The precise nature of the regulatory sequences needed for geneexpression can vary between species or cell types, but in general caninclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. In particular, such 5′ non-transcribed regulatory sequenceswill include a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences can also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA). That heterologous DNA (RNA) is placed underoperable control of transcriptional elements to permit the expression ofthe heterologous DNA in the host cell.

A nucleic acid molecule that encodes the enzyme of the claimed inventioncan be introduced into a cell or cells using methods and techniques thatare standard in the art. For example, nucleic acid molecules can beintroduced by standard protocols such as transformation includingchemical transformation and electroporation, transduction, particlebombardment, etc. Expressing the nucleic acid molecule encoding theenzymes of the claimed invention also may be accomplished by integratingthe nucleic acid molecule into the genome.

Various aspects described herein related to a method, which comprisesrecombinantly expressing in a cell one or more nucleic acids disclosedherein. Some aspects provided herein are directed to cell culture mediumor supernatant collected from culturing a cell expressing one or morenucleic acids described herein. Other aspects provided herein aredirected to a method, comprising culturing in cell culture medium a cellexpressing one or more nucleic acids described herein.

Without wishing to be bound by a theory, microbe-binding moleculescomprising at least a portion of the CRP or or modified versions thereofcan act as broad-spectrum pathogen binding molecules. Accordingly,microbes and/or microbial matter present in a test sample can becaptured or detected using microbe-targeting molecules disclosed hereinwithout identifying the microbe. Since CRP is shown to be bindpreferentially to gram-positive microbes, the molecules, articles,methods, and assays disclosed herein are particularly useful forcapturing, detecting, or clearing gram-positive microbes.

The engineered microbe-targeting molecules can contain sequences fromthe same species or from different species. For example, an interspecieshybrid microbe-targeting molecule can have one of the first domain orsecond domains from a murine species and the other from a human. Theengineered microbe-targeting molecules described herein can also includethose that are made entirely from murine-derived sequences or fullyhuman.

In some embodiments, the microbe-targeting molecule disclosed hereincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 32-38, 40, 41, 43, and 44.

The inventors have discovered inter alia that in microbe-targetingmolecules of formula CRP-X-Fc, where X is a linker, Fc is oriented onthe complement binding face of the CRP pentameter, thereby providingsteric hindrance to complement binding. See FIGS. 7 and 8. Histidine 38is a critical residue for C1q binding and complement activation. TheC-terminus (proline 206) lies very close (<0.6 nm) to this residue and 1nm from other critical residues tyrosine 175 and aspartate 112. Thepresence of the CH2 domain from an immunoglobulin Fc directly fused tothe C-terminus can physically preclude binding of C1q interacting withhistidine 38 and therefore stop binding. When folded, both the C and Ntermini of CRP are located on the complement binding. See FIG. 6.

Without wishing to be bound by a theory, the Fc in the CRP-X-Fcmicrobe-targeting molecule directes the complement binding face of theCRP towards the substrate when the inmicrobe-targeting molecule isconjugated to a substrate. This can provide a second steric hindrancemechanism to reduce complement binding.

Conjugation of Engineered Microbe-Targeting Molecules to a CarrierScaffold

The engineered microbe-targeting molecules can be immobilized orconjugated on any substrate for various applications and/or purposes.For example, when the affinity of a single microbe surface-bindingdomain for a target molecule (e.g., a carbohydrate recognition domainfor a sugar/carbohydrate molecule) is relatively low, and such bindingis generally driven by avidity and multivalency, multivalency of suchengineered microbe-targeting molecules can be effectively increased byattachment of a plurality of the engineered microbe-targeting molecules(e.g., each with one or two or more carbohydrate recognition domains) toa solid substrate (e.g., a nanometer- or micrometer-sized bead) at ahigh density, which can be varied to provide optimal functionality.Alternatively, the engineered microbe-targeting molecules can beimmobilized on a solid substrate for easy handling during usage, e.g.,for isolation, observation or microscopic imaging.

Accordingly, a further aspect provided herein is an article or productfor targeting or binding microbes comprising at least one, including atleast two, at least three, at least four, at least five, at least ten,at least 25, at least 50, at least 100, at least 250, at least 500, ormore engineered microbe-targeting molecules conjugated to a carrierscaffold or a surface thereof. The “carrier scaffold” is also referredto as a “carrier substrate” herein. In some embodiments, surface of thecarrier scaffold can be coated with the microbe-targeting moleculedisclosed herein. As used herein, the term “article” refers to anydistinct physical microscale or macroscale object. An article comprisinga microbe-targeting molecule conjugated to a carrier scaffold is alsoreferred to as a “microbe-binding article” or a “microbe-targetingarticle” herein.

Without limitations, the carrier scaffold can be selected from a widevariety of materials and in a variety of formats. For example, thecarrier scaffold can be utilized in the form of beads or particles(including nanoparticles, microparticles, polymer microbeads, magneticmicrobeads, and the like), filters, fibers, screens, mesh, tubes, hollowfibers, scaffolds, plates, channels, gold particles, magnetic materials,planar shapes (such as a rectangular strip or a circular disk, or acurved surface such as a stick), other substrates commonly utilized inassay formats, and any combinations thereof.

Examples of carrier scaffolds include, but are not limited to, nucleicacid scaffolds, protein scaffolds, lipid scaffolds, dendrimers,microparticles or microbeads, nanotubes, microtiter plates, medicalapparatuses (e.g., needles or catheters) or implants, dipsticks or teststrips, microchips, filtration devices or membranes, membranes,diagnostic strips, hollow-fiber reactors, microfluidic devices, livingcells and biological tissues or organs, extracorporeal devices, mixingelements (e.g., spiral mixers), and the like. In some embodiments, thecarrier scaffold can be in the form of a continuous roll on which thetest area(s) and optionally reference area(s) are present in the form ofcontinuous lines or a series of spots.

The carrier scaffold can be made of any material, including, but notlimited to, metal, metal alloy, polymer, plastic, paper, glass, fabric,packaging material, biological material such as cells, tissues,hydrogels, proteins, peptides, nucleic acids, and any combinationsthereof.

In some embodiments, the microbe-targeting articles disclosed herein canbe used to catpture, detect, or remove microbe contaminants from anysource or in any fluid, e.g., a biological fluid (e.g., blood sample),environmental fluid or surface (e.g., wastewater, building or machinesurface), or an edible substrance or fluid (e.g., food, water). In someembodiments where the fluid is blood, after removal of themicrobe/pathogen from the blood collected from a subject with themicrobe-targeting magnetic microbeads, the blood can be circulated backto the same subject as a therapeutic intervention. In some embodiments,the microbe-targeting articles disclosed herein can be used indiagnostics as a means of collecting potential pathogens foridentification; not only in the diagnosis of disease, but in theidentification of water- or food-borne pathogens, particulates or othercontaminants. Alternatively, the carrier scaffold can comprise ahollow-fiber reactor or any other blood filtration membrane or flowdevice (e.g., a simple dialysis tube, spiral mixer or static mixer) orother resins, fibers, or sheets to selective bind and sequester thebiological pathogens.

The microbe-binding articles disclosed herein also can be used aspoint-of-care diagnostic tools for microbe or pathogen detection. By wayof example only, a microbe-binding article can be brought into contactwith a test sample (e.g., a blood sample) from a patient or a subject,and incubated for a period of time, e.g., at least about 15 seconds, atleast about 30 seconds, at least about 1 min, at least about 2 mins, atleast about 5 mins, at least about 10 mins, at least about 15 mins, atleast about 30 mins, at least about 1 hour or more. In some embodiments,the incubated dipstick or test strip can then be incubated in a blockingagent (e.g., BSA, normal serum, casesin, non-fat dry milk, and/or anycommercially-available blocking agents to minimize non-specificbinding). Depending on different embodiments of the engineeredmicrobe-targeting molecules, in some embodiments, the microbe-bindingdipstick or test strip after contact with a test sample (e.g., a bloodsample) can be further contacted with at least one additional agent tofacilitate detection of pathogen, and/or to increase specificity of thepathogen detection. For example, some embodiments of the dipstick ortest strip after contact with a test sample (e.g., a blood sample) canbe further contacted with a detectable label that is conjugated to amolecule that binds to a microbe and/or microbial matter. Examples ofsuch molecules can include, but are not limited to, one or moreembodiments of the engineered microbe-targeting molecule describedherein, an antibody specific for the microbes or pathogens to bedetected, a protein, a peptide, a carbohydrate or a nucleic acid that isrecognized by the microbes or pathogens to be detected, and anycombinations thereof.

In some embodiments, the readout of the microbe-binding article can beperformed in a system or device, e.g., a portable device. The system ordevice can display a signal indicating the presence or the absence of amicrobial infection in a test sample, and/or the extent of the microbialinfection.

The particular format or material of the carrier scaffold depends on theparticular use or application, for example, the separation/detectionmethods employed in an assay application. In some embodiments, theformat or material of the carrier scaffold can be chosen or modified tomaximize signal-to-noise ratios, e.g., to minimize background binding orfor ease of separation of reagents and cost. For example, carrierscaffold can be treated or modified with surface chemistry to minimizechemical agglutination and non-specific binding. In some embodiments, atleast a portion of the carrier scaffold surface that is in contact witha test sample can be treated to become less adhesive to any molecules(including microbes, if any) present in a test sample. By way of exampleonly, the carrier scaffold surface in contact with a test sample can besilanized or coated with a polymer such that the surface is inert to themolecules present in the test sample, including but not limited to,cells or fragments thereof (including blood cells and blood components),proteins, nucleic acids, peptides, small molecules, therapeutic agents,microbes, microorganisms and any combinations thereof. In otherembodiments, a carrier scaffold surface can be treated with anomniphobic layer, which can allow binding of a microbe by the engineeredmicrobe-targeting molecule without a subsequent hydrophobic bindingbetween the microbe and the carrier scaffold surface. See, e.g., Wong TS et al., “Bioinspired self-repairing slippery surfaces withpressure-stable omniphobicity.” (2011) Nature 477 (7365): 443-447, andInternational Application No.: PCT/US12/21928, the content of which isincorporated herein by reference, for methods to produce a slipperycarrier scaffold surface. Accordingly, non-specific binding of moleculesfrom the test sample (including microbes and/or microbial matter) to asubstrate surface can be reduced, thus increasing the sensitivity of themicrobial detection.

In some embodiments, the carrier scaffd can be fabricated from or coatedwith a biocompatible material. As used herein, the term “biocompatiblematerial” refers to any material that does not deteriorate appreciablyand does not induce a significant immune response or deleterious tissuereaction, e.g., toxic reaction or significant irritation, over time whenimplanted into or placed adjacent to the biological tissue of a subject,or induce blood clotting or coagulation when it comes in contact withblood. Suitable biocompatible materials include, for example,derivatives and copolymers of polyimides, poly(ethylene glycol),polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylates,polyamides, polyesters, polycarbonates, and polystyrenes. In someembodiments, biocompatible materials can include metals, such astitanium and stainless steel, or any biocompatible metal used in medicalimplants. In some embodiments, biocompatible materials can include papersubstrate, e.g., as a carrier scaffold for a diagnostic strip. In someembodiments, biocompatible materials can include peptides or nucleicacid molecules, e.g., a nucleic acid scaffold such as a 2-D DNA sheet or3-D DNA scaffold.

Additional material that can be used to fabricate or coat a carrierscaffold include, without limitations, polydimethylsiloxane, polyimide,polyethylene terephthalate, polymethylmethacrylate, polyurethane,polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon,polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene,polyacrylonitrile, polybutadiene, poly(butylene terephthalate),poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol),styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinylbutyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and anycombination thereof.

In some embodiments, the carrier scaffd can be fabricated from or coatedwith a biodegradable material. As used herein, the term “biodegradable”refers to the ability of a composition to erode or degrade in vivo toform smaller chemical fragments. Degradation can occur, for example, byenzymatic, chemical or physical processes. Non-limiting examples ofbiodegradable polymers that can be used in aspects provided hereininclude poly(lactide)s, poly(glycolide)s, poly(lactic acid)s,poly(glycolic acid)s, poly (lactide-co-glycolide), polyanhydrides,polyorthoesters, polycaprolactone, polyesteramides, polycarbonate,polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymersthereof.

Other additional biodegradable polymers include biodegradablepolyetherester copolymers. Generally speaking, the polyetherestercopolymers are amphiphilic block copolymers that include hydrophilic(for example, a polyalkylene glycol, such as polyethylene glycol) andhydrophobic blocks (for example, polyethylene terephthalate). Anexemplary block copolymer is, but is not limited to, poly(ethyleneglycol)-based and poly(butylene terephthalate)-based blocks (PEG/PBTpolymer). PEG/PBT polymers are commercially available from OctoPlus Inc,under the trade designation PolyActive™. Non-limiting examples ofbiodegradable copolymers or multiblock copolymers include the onesdescribed in U.S. Pat. Nos. 5,980,948 and 5,252,701, the contents ofwhich are incorporated herein by reference.

Other biodegradable polymer materials include biodegradableterephthalate copolymers that include a phosphorus-containing linkage.Polymers having phosphoester linkages, called poly(phosphates),poly(phosphonates) and poly(phosphites), are known in the art. See, forexample, Penczek et al., Handbook of Polymer Synthesis, Chapter 17:“Phosphorus-Containing Polymers,” 1077-1 132 (Hans R. Kricheldorf ed.,1992), as well as U.S. Pat. Nos. 6,153,212; 6,485,737; 6,322,797;6,600,010; 6,419,709; 6,419,709; 6,485,737; 6,153,212; 6,322,797 and6,600,010, the contents of which are incorporated herein by reference.

Biodegradable polyhydric alcohol esters can also be used as a materialof a carrier scaffold (e.g., a microparticle) (See U.S. Pat. No.6,592,895, which is incorporated herein by reference). In someembodiments, the biodegradable polymer can be a three-dimensionalcrosslinked polymer network containing hydrophobic and hydrophiliccomponents which forms a hydrogel with a crosslinked polymer structure,such as the one described in U.S. Pat. No. 6,583,219. In yet furtherembodiments, the biodegradable polymer can comprise a polymer based upona-amino acids (such as elastomeric copolyester amides or copolyesterurethanes, as described in U.S. Pat. No. 6,503,538, which isincorporated herein by reference).

In some embodiments, the carrier scaffold can comprise a paper,nitrocellulose, glass, plastic, polymer, membrane material, nylon, andany combinations thereof. This is useful for using the article as a teststrip of a dipstick.

As used herein, by the “coating” or “coated” is generally meant a layerof molecules or material formed on an outermost or exposed layer of asurface. With respect to a coating of engineered microbe-targetingmolecules on a carrier scaffold, the term “coating” or “coated” refersto a layer of engineered microbe-targeting molecules formed on anoutermost or exposed layer of a carrier scaffold surface. In someembodiments, the carrier scaffold surface can encompass an outer surfaceor an inner surface, e.g., with respect to a hollow structure. Forexample, the inner surface of a needle or catheter can be coated withthe engineered microbe-targeting molecules described herein. This can beuseful for removing any potential microbe contaminants from a fluidbefore administering the fluid to a subject.

The amount of the engineered microbe-targeting molecules conjugated toor coating on a carrier scaffold can vary with a number of factors suchas a surface area, conjugation/coating density, types of engineeredmicrobe-targeting molecules, and/or binding performance. A skilledartisan can determine the optimum density of engineeredmicrobe-targeting molecules on a carrier scaffold using any methodsknown in the art. By way of example only, for magnetic microparticles asa carrier scaffold (as discussed in detail later), the amount of theengineered microbe-targeting molecules used for conjugating to orcoating magnetic microparticles can vary from about 1 wt % to about 30wt %, or from about 5 wt % to about 20 wt %. In some embodiments, theamount of the engineered microbe-targeting molecules used forconjugating to or coating magnetic microparticles can be higher orlower, depending on a specific need. However, it should be noted that ifthe amount of the engineered microbe-targeting molecules used forconjugating to or coating the magnetic microparticles is too low, themagnetic microparticles can show a lower binding performance with apathogen/microbe. On the contrary, if the amount of the engineeredmicrobe-targeting molecules used for conjugating to or coating themagnetic microparticles is too high, the dense layer of the engineeredmicrobe-targeting molecules can exert an adverse influence on themagnetic properties of the magnetic microbeads, which in turn candegrade the efficiency of separating the magnetic microbeads from afluid utilizing the magnetic field gradient.

In some embodiments, the carrier scaffold can further comprise at leastone area adapted for use as a reference area. By way of example only,the reference area can be adapted for use as a positive control,negative control, a reference, or any combination thereof. In someembodiments, the carrier scaffold can further comprise at least twoareas, wherein one area is adapted for a positive control and the secondarea is adapated for a negative control.

In some embodiments, the carrier scaffold can further comprise at leastone reference area or control area for comparison with a readout signaldetermined from the test area. The reference area generally excludes theengineered microbe-targeting molecules, e.g., to account for anybackground signal. In some embodiments, the reference area can includeone or more known amounts of the detectable label that the engineeredmicrobe-targeting molecules in the test area encompass. In suchembodiments, the reference area can be used for calibration such thatthe amount of microbes in a test sample can be estimated or quantified.

In some embodiments, the carrier scaffold further at least one secondmicrobe-targeting molecule, wherein the second microbe-targetingmolecule comprises at least one first domain wherein the CRP domain isreplaced by a microbe-binding domain of microbe-binding domain proteinwhich is not CRP. Thus, the second microbe-targeting molecule comprisesat least one first domain comprising at least a portion of amicrobe-binding domain of a microbe-binding protein, wherein themicrobe-binding protein is not a CRP; a second domain, as described inthis disclosure; and a linker conjugating the first and the seconddomains. Microbe-binding domains that do not comprise CRP are describedelsewhere in the disclosure. Exemplary second microbe-targetingmolecules are described, for example, in PCT Application No.PCT/US2011/021603 filed Jan. 19, 2011 and No. PCT/US2012/047201, filedJul. 18, 2012, and U.S. Provisional Application No. 61/691,983 filedAug. 22, 2012, contents of all of which are incorporated herein byreference in their entireties. In some embodiments, the secondmicrobe-targeting molecule comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 9, 11, 12, and 23-27.

In some embodiments, the carrier scaffold can further comprise adetectable label. The detetable label can be separate from themicrobe-targeting molecules conjugated with the carrier scaffold orlinked to the the microbe-targeting molecules conjugated with thecarrier scaffold.

Microbe-targeting microparticles: In some embodiments, the carrierscaffold is a microparticle. Accordingly, some embodiments describedherein provide a microbe-targeting microparticle comprising at least oneengineered microbe-targeting molecule on its surface. The term“microparticle” as used herein refers to a particle having a particlesize of about 0.001 μm to about 1000 μm, about 0.005 μm to about 50 μm,about 0.01 μm to about 25 μm, about 0.05 μm to about 10 μm, or about0.05 μm to about 5 μm. In one embodiment, the microparticle has aparticle size of about 0.05 μm to about 1 μm. In one embodiment, themicroparticle is about 0.09 μm-about 0.2 μm in size.

In some embodiments, the microparticle can range in size from 1 nm to 1mm, about 2.5 nm to about 500 μm, or about 5 nm to about 250 μm in size.In some embodiments, microparticle can be about 5 nm to about 100 μm insize. In some embodiments, microparticle can be about 0.01 μm to about10 μm in size. In some embodiments, the microparticle can be about 0.05μm to about 5 μm in size. In some embodiments, the microparticle can beabout 0.08 μm to about 1 μm in size. In one embodiment, themicroparticle can be about 10 nm to about 10 μm in size. In someembodiments, the the microparticle can be about 1 nm to about 1000 nm,from about 10 nm to about 500 nm, from about 25 nm to about 300 nm, fromabout 40 nm to about 250 nm, or from about 50 nm to about 200 nm. In oneembodiment, the microparticle can be about 50 nm to about 200 nm.

It will be understood by one of ordinary skill in the art thatmicroparticles usually exhibit a distribution of particle sizes aroundthe indicated “size.” Unless otherwise stated, the term “size” as usedherein refers to the mode of a size distribution of microparticles,i.e., the value that occurs most frequently in the size distribution.Methods for measuring the microparticle size are known to a skilledartisan, e.g., by dynamic light scattering (such as photocorrelationspectroscopy, laser diffraction, low-angle laser light scattering(LALLS), and medium-angle laser light scattering (MALLS)), lightobscuration methods (such as Coulter analysis method), or othertechniques (such as rheology, and light or electron microscopy).

Without limitations, the microparticle can be of any shape. Thus, themicroparticle can be, but is not limited to, spherical, rod, elliptical,cylindrical, disc, and the like. In some embodiments, the term“microparticle” as used herein can encompass a microsphere. The term“microsphere” as used herein refers to a microparticle having asubstantially spherical form. A substantially spherical microparticle isa microparticle with a difference between the smallest radii and thelargest radii generally not greater than about 40% of the smaller radii,and more typically less than about 30%, or less than 20%.

In some embodiments, the microparticles having a substantially sphericalshape and defined surface chemistry can be used to minimize chemicalagglutination and non-specific binding.

In one embodiment, the term “microparticle” as used herein encompasses amicrocapsule. The term “microcapsule” as used herein refers to amicroscopic capsule that contains an active ingredient, e.g., atherapeutic agent or an imagining agent. Accordingly, in someembodiments, the microparticles comprising on their surface engineeredmicrobe-targeting molecules can encapsulate at least one activeingredient therein, e.g., a therapeutic agent to treat an infection, andbe used as a cell-targeted drug delivery device. In such embodiments,the microparticles can comprise biocompatible polymers as describedherein. In some embodiments, the microparticles can further comprisebiodegradable polymers, e.g., for releasing the encapsulated drugs.

In general, any biocompatible material well known in the art forfabrication of microparticles can be used in embodiments of themicroparticle described herein. Accordingly, a microparticle comprisinga lipidic microparticle core is also within the scope described herein.An exemplary lipidic microparticle core is, but is not limited to, aliposome. A liposome is generally defined as a particle comprising oneor more lipid bilayers enclosing an interior, e.g., an aqueous interior.In one embodiment, a liposome can be a vesicle formed by a bilayer lipidmembrane. Methods for the preparation of liposomes are well described inthe art, e.g., Szoka and Papahadjopoulos (1980) Ann. Rev. Biophys.Bioeng. 9: 467, Deamer and Uster (1983) Pp. 27-51 In: Liposomes, ed. M.J. Ostro, Marcel Dekker, New York.

Microbe-targeting magnetic microparticles: In some embodiments, themicroparticle is a magnetic microparticle. Thus, in some embodiments,provided herein is a “microbe-targeting magnetic microparticle” whereina magnetic microparticle comprising on its surface at least oneengineered microbe-targeting molecule. Without limitations, suchmicrobe-targeting magnetic microparticles can be used to separatemicrobes or pathogens from a test sample, e.g., but not limited to, anyfluid, including a biological fluid such as blood. In some embodiments,the microbe-targeting magnetic microparticle can be used to removeliving microbes or pathogens. Using magnetic microparticles as asubstrate can be advantageous because the microbe-bound magneticmicroparticles can be easily separated from a sample fluid using amagnetic field gradient, be examined for the presence of the microbe,and/or be used to transfer the collected microbes to conventionalpathogen culture and sensitivity testing assays. Thus, in someembodiments, the microbe-targeting magnetic microparticles can be usedto capture, detect, or remove microbe contaminants from any source or inany fluid, e.g., a biological fluid (e.g., blood sample), environmentalfluid or surface (e.g., wastewater, building or machine surface), or anedible substrance or fluid (e.g., food, water). In some embodimentswhere the fluid is blood, after removal of the microbe/pathogen from theblood collected from a subject with the microbe-targeting magneticmicrobeads, the blood can be circulated back to the same subject as atherapeutic intervention. In some embodiments, the microbe-targetingmagnetic microbeads can be used in diagnostics as a means of collectingpotential pathogens for identification; not only in the diagnosis ofdisease, but in the identification of water- or food-borne pathogens,particulates or other contaminants. Alternatively, the solid substratecan comprise a hollow-fiber reactor or any other blood filtrationmembrane or flow device (e.g., a simple dialysis tube, spiral mixer orstatic mixer) or other resins, fibers, or sheets to selective bind andsequester the biological pathogens.

Magnetic microparticles can be manipulated using magnetic field ormagnetic field gradient. Such particles commonly consist of magneticelements such as iron, nickel and cobalt and their oxide compounds.Magnetic microparticles are well-known and methods for their preparationhave been described in the art. See, e.g., U.S. Pat. Nos. 6,878,445;5,543,158; 5,578,325; 6,676,729; 6,045,925; and 7,462,446; and U.S.Patent Publications No. 2005/0025971; No. 2005/0200438; No.2005/0201941; No. 2005/0271745; No. 2006/0228551; No. 2006/0233712; No.2007/01666232; and No. 2007/0264199, the contents of which areincorporated herein by reference.

Magnetic microparticles are also widely and commercially available, withor without functional groups capable of conjugation with themicrobe-targeting molecules disclosed herein. Magnetic microparticlesfunctionalized with various functional groups, e.g., amino groups,carboxylic acid groups, epoxy groups, tosyl groups, or silica-likegroups, are also widely and commercially available. Suitable magneticmicroparticles are commercially available such as from AdemTech,Miltenyi, PerSeptive Diagnostics, Inc. (Cambridge, Mass.); InvitrogenCorp. (Carlsbad, Calif.); Cortex Biochem Inc. (San Leandro, Calif.); andBangs Laboratories (Fishers, Ind.). In particular embodiments, magneticmicroparticles that can be used herein can be any DYNABEADS® magneticmicrobeads (Invitrogen Inc.), depending on the substrate surfacechemistry.

Microbe-targeting cells: In some embodiments, the carrier scaffold towhich the engineered microbe-targeting molecule binds can be a livingcell, or a biological tissue or organ. For example, the living cells canbe associated with an immune response, and such cells include, but arenot limited to, a phagocyte (macrophage, neutrophil, and dendriticcell), mast cell, eosinophil, basophil, and/or natural killer cell.Alternatively, the living cell can be the cell of biological tissues ororgans of the immune system, such as spleen, lymph nodes, lymphaticvessels, tonsils, thymus, bone marrow, Peyer's patches, connectivetissues, mucous membranes, the reticuloendothelial system, etc. In someembodiments, the surface to which the engineered microbe-targetingmolecules bind can also be the extracellular matrix of one or more ofthese tissues or organs.

Microbe-binding microtiter plates: In some embodiments, the bottomsurface of microtiter wells can be coated with the engineeredmicrobe-targeting molecules described herein, e.g., for detecting and/ordetermining the amount of microbes in a sample. After microbes orpathogens in the sample binding to the engineered microbe-targetingmolecules bound to the microwell surface, the rest of the sample can beremoved. Detectable molecules that can also bind to microbes orpathogens (e.g., an engineered microbe-targeting molecule conjugated toa detectable molecule as described herein) can then be added to themicrowells with microbes/pathogens for detection of microbes/pathogens.Various signal detection methods for determining the amount of proteins,e.g., using enzyme-linked immunosorbent assay (ELISA), with differentdetectable molecules have been well established in the art, and thosesignal detection methods can also be employed herein to facilitatedetection of the signal induced by microbes/pathogens binding on theengineered microbe-targeting molecules.

Microbe-binding dipsticks/test strips: In some embodiments, the carrierscaffold having the microbe-targeting molecule conjugated thereon can bein the form of a dipstick and/or a test strip for capture, detection, orclearance of microbes or pathogens. For example, a dipstick and/or atest strip can include at least one test area containing one or moreengineered microbe-targeting molecules described herein. The dipstickand/or a test strip can be in any shape and/or in any format, e.g., aplanar shape such as a rectangular strip or a circular disk, or a curvedsurface such as a stick. Alternatively, a continuous roll can beutilized, rather than discrete test strips, on which the test area(s)and optionally reference area(s) are present in the form of continuouslines or a series of spots. In some embodiments, the microbe-bindingdipsticks or test strips described herein can be used as point-of-carediagnostic tools for microbe or pathogen detection.

In some embodiments, the carrier scaffold in the form of a dipstick or atest strip can be made of any material, including, without limitations,paper, nitrocellulose, glass, plastic, polymer, membrane material,nylon, and any combinations thereof. In one embodiment, the carrierscaffold in the form of a dipstick or a test strip can include paper. Inone embodiment, the carrier scaffold in the form of a dipstick or a teststrip can include nylon.

In some embodiments, the dipstick or a test strip can further compriseat least one reference area or control area for comparison with areadout signal determined from the test area. The reference areagenerally excludes the engineered microbe-targeting molecules, e.g., toaccount for any background signal. In some embodiments, the referencearea can include one or more known amounts of the detectable label thatthe engineered microbe-targeting molecules in the test area encompass.In such embodiments, the reference area can be used for calibration suchthat the amount of microbes in a test sample can be estimated orquantified.

In some embodiments, the dipstick/test strip can further comprise adetectable label as described herein. The detectable label can be linkedto the microbe-targeting molecule conjugated with the dipstick/teststrip or separate from the microbe-targeting molecule conjugated withthe dipstick/test strip.

In one embodiment, about 1 μg to about 100 μg microbe-binding moleculescan be coated on or attached to a dipstick or membrane surface. Inanother embodiment, about 3 μg to about 60 μg microbe-binding moleculescan be coated on or attached to a dipstick or membrane surface. In someembodiments, about 0.1 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 5 mg/mL to about 20mg/mL microbe-binding molecules can be coated on or attached to adipstick or membrane surface. In one embodiment, about 11.5 mg/mLmicrobe-binding molecules can be coated on or attached to a dipstick ormembrane surface.

Exemplary Process for Capture/Detection of a Microbe or Microbial Matterin a Test Sample

An exemplary process for detecting a microbe and/or microbial matter ina test sample is described herein. As shown in FIG. 1, the process 1200comprises the optional step 1202 (preprocessing of the sample), step1204 (processing of the sample), step 1206 comprising 1208 (microbecapture) and 1210 (microbe separation), and 1212 (microbe detection).While these are discussed as discrete processes, one or more of thepreprocessing, processing, capture, microbe separation, and detectioncan be performed in a microfluidic device. Use of a microfluidic devicecan automate the analysis process and/or allow analysis of multiplesamples at the same time. One of skill in the art is well aware ofmethods in the art for collecting, handling and processing biologicalfluids which can be used in the practice of the present disclosure. Theprocess described herein can allow sample analysis at in short timeperiods. For example, the process can be completed in less than 6 hours,less than 5 hours, less than 4 hours, less than 3 hours, less than 2hours, less than 1 hour, less than 30 minutes. In some embodiments,presence and identity of a microbe in the sample can be done within 10minutes to 60 minutes of starting the process.

In some embodiments, the sample can be a biological fluid, e.g., blood,plasma, serum, lactation products, amniotic fluids, sputum, saliva,urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration,mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears,tracheal aspirate, and any mixtures thereof. For example, the sample canbe a whole blood sample obtained from a subject.

The process described herein can be utilized to detect the presence of amicrobe in a sample of any given volume. In some embodiments, samplevolume is about 0.25 ml to about 50 ml, about 0.5 ml to about 25 ml,about 1 ml to about 15 ml, about 2 ml to about 10 ml. In someembodiments, sample volume is about 5 ml. In one embodiment, samplevolume is about 5 ml to abut 10 ml.

1202 (Sample preprocessing): It can be necessary or desired that a testsample, such as whole blood, be preprocessed prior to microbe detectionas described herein, e.g., with a preprocessing reagent. Even in caseswhere pretreatment is not necessary, preprocessing can be optionallydone for mere convenience (e.g., as part of a regimen on a commercialplatform). A preprocessing reagent can be any reagent appropriate foruse with the assays or processes described herein.

The sample preprocessing step generally comprises adding one or morereagent to the sample. This preprocessing can serve a number ofdifferent purposes, including, but not limited to, hemolyzing bloodcells, dilution of sample, etc. The preprocessing reagents can bepresent in the sample container before sample is added to the samplecontainer or the preprocessing reagents can be added to a sample alreadypresent in the sample container. When the sample is a biological fluid,the sample container can be a VACUTAINER®, e.g., a heparinizedVACUTAINER®.

The preprocessing reagents include, but are not limited to, surfactantsand detergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases and the like), and solvents, such as buffersolutions. In some embodiments, a preprocessing reagent is a surfactantor a detergent. In one embodiment, the preprocessing reagent is TRITONX100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol).

Amount of preprocessing reagent to be added can depend on a number offactors. Generally, the preprocessing reagent is added to a finalconcentration of about 0.1 mM to about 10 mM. If a liquid, thepreprocessing reagent can be added so as to dilute the sample at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 60%, at least 80%, at least 90%, at least1-fold, at least 2-fold, at least 3-fold, or at least 5-fold.

After addition of the preprocessing reagent, the reagent can be mixedinto the sample. This can be simply accomplished by agitating thesample, e.g., shaking or vortexing the sample and/or moving the samplearound, if it is in a microfluidic device.

After addition of the preprocessing reagent, the sample mixture can beincubated for a period of time. For example, the sample mixture can beincubated for at least one minute, at least two minutes, at least threeminutes, at least four minutes, at least five minutes, at least tenminutes, at least fifteen minutes, at least thirty minutes, at leastforty-five minutes, or at least one hour. In some embodiments,incubation is for about 5 seconds to about 60 seconds. In someembodiments, incubation is for about 10 to about 20 minutes. In oneembodiment, incubation is for about 15 minutes. In some embodiments,there is no incubation and the sample mixture is used directly in thesample processing step.

Without limitations, incubation can be at any appropriate temperature.For example, the incubation can be at room temperature (about 16° C. toabout 30° C.), a cold temperature (about 16° C. or lower, e.g., fromabout −4° C. to about 16° C.), or an elevated temperature (about 30° C.or higher, e.g., about 25° C. to about 95° C.). In some embodiments, thesample is incubated for about fifteen minutes at room temperature.

1204 (Sample processing): After the optional preprocessing step, thesample can be optionally processed by adding one or more processingreagents to the sample. These processing reagents can serve to lysecells, degrade unwanted molecules present in the sample and/or dilutesample for further processing. These processing reagents include, butare not limited to, surfactants and detergents, salts, cell lysingreagents, anticoagulants, degradative enzymes (e.g., proteases, lipases,nucleases, lipase, collagenase, cellulases, amylases and the like), andsolvents, such as buffer solutions. Amount of the processing reagent tobe added can depend on the particular sample to be analyzed, the timerequired for the sample analysis, identity of the microbe to be detectedor the amount of microbe present in the sample to be analyzed.

It is not necessary, but if one or more reagents are to be added theycan present in a mixture (e.g., in a solution, “processing buffer”) inthe appropriate concentrations. Amount of the various components of theprocessing buffer can vary depending upon the sample, microbe to bedetected, concentration of the microbe in the sample, or time limitationfor analysis.

Generally, addition of the processing buffer can increase the volume ofthe sample by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.In some embodiments, about 50 μl to about 5000 μl of the processingbuffer are added for each ml of the sample. In some embodiments, about100 μl to about 250 μl of the processing buffer are added for each ml ofthe sample. In one embodiment, about 800 μl of the processing buffer areadded for each 200 μl of the sample.

In some embodiments, a detergent or surfactant comprises about 5% toabout 20% of the processing buffer volume. In some embodiment, adetergent or surfactant comprises about 5% to about 15% of theprocessing buffer volume. In one embodiment, a detergent or surfactantcomprises about 10% of the processing buffer volume.

Exemplary surfactants and detergents include, but are not limited to,sulfates, such as, ammonium lauryl sulfate, sodium dodecyl sulfate(SDS), and sodium lauryl ether sulfate (SLES) sodium myreth sulfate;sulfonates, such as, dioctyl sodium sulfosuccinate (Docusates),perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, alkyl benzenesulfonates, and3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); phosphates, such as alkyl aryl ether phosphate and alkyl etherphosphate; carboxylates, such as fatty acid salts, sodium stearate,sodium lauroyl sarcosinate, perfluorononanoate, and perfluorooctanoate(PFOA or PFO); octenidine dihydrochloride; alkyltrimethylammonium salts,such as cetyl trimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC);polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; dioctadecyldimethylammoniumbromide (DODAB); sultaines, such as cocamidopropyl hydroxysultaine;cetyl alcohol; stearyl alcohol; cetostearyl alcohol (consistingpredominantly of cetyl and stearyl alcohols); oleyl alcohol;polyoxyethylene glycol alkyl ethers (Brij) such as, octaethylene glycolmonododecyl ether and pentaethylene glycol monododecyl ether;polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such asdecyl glucoside, lauryl glucoside and octyl glucoside; polyoxyethyleneglycol octylphenol ethers, such as TRITON X-100(2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol); polyoxyethyleneglycol alkylphenol ethers, such as Nonoxynol-9; glycerol alkyl esters,such as glyceryl laurate; polyoxyethylene glycol sorbitan alkyl esters,such as Polysorbate 20 (Polyoxyethylene (20) sorbitan monolaurate),Polysorbate 40 (Polyoxyethylene (20) sorbitan monopalmitate),Polysorbate 60 (Polyoxyethylene (20) sorbitan monostearate), andPolysorbate 80 (Polyoxyethylene (20) sorbitan monooleate); cocamide ME;cocamide DEA; dodecyldimethylamine oxide; poloxamers; DOC; nonylphenoxypolyethoxylethanol NP-40 (Tergitol-type NP-40); octylphenoxypolyethoxylethanol (Noidet P-40); cetyltrimethylammonium bromide;and any mixtures thereof.

In some embodiments, one ml of the processing buffer can comprise about0.1 U to about 100 U of a degradative enzyme. In some embodiments, oneml of the processing buffer comprises about 5 U to about 50 U of adegradative enzyme. In one embodiment, one ml of the processing buffercomprises about 10 U of a degradative enzyme. Enzyme unit (U) is an artknown term for the amount of a particular enzyme that catalyzes theconversion of 1 μmmol of substrate per minute.

In some embodiments, one ml of the processing buffer can comprise about1 mg to about 10 μg of an anti-coagulant. In some embodiment, one ml ofthe processing buffer can comprise about 1 μg to about 5 μg of ananti-coagulant. In one embodiment, one ml of the processing buffercomprises about 4.6 μg of an anti-coagulant.

In some embodiments, one ml of the processing buffer can comprise about1 mg to about 10 mg of anti-coagulant. In some embodiment, one ml of theprocessing buffer can comprise about 1 mg to about 5 mg ofanti-coagulant. In one embodiment, one ml of the processing buffercomprises about 4.6 mg of anti-coagulant.

Exemplary anti-coagulants include, but are not limited to, heparin,heparin substitutes, salicylic acid, D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone (PPACK), Hirudin, Ancrod (snake venom, Vipronax),tissue plasminogen activator (tPA), urokinase, streptokinase, plasmin,prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors,dextrans, thrombin antagonists/inhibitors, ethylene diamine tetraaceticacid (EDTA), acid citrate dextrose (ACD), sodium citrate, citratephosphate dextrose (CPD), sodium fluoride, sodium oxalate, potassiumoxalate, lithium oxalate, sodium iodoacetate, lithium iodoacetate andmixtures thereof.

Suitable heparinic anticoagulants include heparins or active fragmentsand fractions thereof from natural, synthetic, or biosynthetic sources.Examples of heparin and heparin substitutes include, but are not limitedto, heparin calcium, such as calciparin; heparin low-molecular weight,such as enoxaparin and lovenox; heparin sodium, such as heparin,lipo-hepin, liquaemin sodium, and panheprin; heparin sodiumdihydroergotamine mesylate; lithium heparin; and ammonium heparin.

Suitable prothrombopenic anticoagulants include, but are not limited to,anisindione, dicumarol, warfarin sodium, and the like.

Examples of phosphodiesterase inhibitors suitable for use hereininclude, but are not limited to, anagrelide, dipyridamole,pentoxifyllin, and theophylline.

Suitable dextrans include, but are not limited to, dextran70, such asHYSKON™ (CooperSurgical, Inc., Shelton, Conn., U.S.A.) and MACRODEX™(Pharmalink, Inc., Upplands Vasby, Sweden), and dextran 75, such asGENTRAN™ 75 (Baxter Healthcare Corporation).

Suitable thrombin antagonists include, but are not limited to, hirudin,bivalirudin, lepirudin, desirudin, argatroban, melagatran, ximelagatranand dabigatran.

As used herein, anticoagulants can also include factor Xa inhibitors,factor Ha inhibitors, and mixtures thereof. Various direct factor Xainhibitors are known in the art including, those described in Hirsh andWeitz, Lancet, 93:203-241, (1999); Nagahara et al. Drugs of the Future,20: 564-566, (1995); Pinto et al, 44: 566-578, (2001); Pruitt et al,Biorg. Med. Chem. Lett., 10: 685-689, (2000); Quan et al, J. Med. Chem.42: 2752-2759, (1999); Sato et al, Eur. J. Pharmacol, 347: 231-236,(1998); Wong et al, J. Pharmacol. Exp. Therapy, 292:351-357, (2000).Exemplary factor Xa inhibitors include, but are not limited to,DX-9065a, RPR-120844, BX-807834 and SEL series Xa inhibitors. DX-9065ais a synthetic, non-peptide, propanoic acid derivative, 571 D selectivefactor Xa inhibitor. It directly inhibits factor Xa in a competitivemanner with an inhibition constant in the nanomolar range. See forexample, Herbert et al, J. Pharmacol. Exp. Ther. 276:1030-1038 (1996)and Nagahara et al, Eur. J. Med. Chem. 30(suppl):140s-143s (1995). As anon-peptide, synthetic factor Xa inhibitor, RPR-120844 (Rhone-PoulencRorer), is one of a series of novel inhibitors which incorporate3-(S)-amino-2-pyrrolidinone as a central template. The SEL series ofnovel factor Xa inhibitors (SEL1915, SEL-2219, SEL-2489, SEL-2711:Selectide) are pentapeptides based on L-amino acids produced bycombinatorial chemistry. They are highly selective for factor Xa andpotency in the pM range.

Factor Ha inhibitors include DUP714, hirulog, hirudin, melgatran andcombinations thereof. Melagatran, the active form of pro-drugximelagatran as described in Hirsh and Weitz, Lancet, 93:203-241, (1999)and Fareed et al. Current Opinion in Cardiovascular, pulmonary and renalinvestigational drugs, 1:40-55, (1999).

Generally, salt concentration of the processing buffer can range fromabout 10 mM to about 100 mM. In some embodiments, the processing buffercomprises a salt at a concentration of about 25 mM to about 75 mM. Insome embodiment, the processing buffer comprises a salt at aconcentration of about 45 mM to about 55 mM. In one embodiment, theprocessing buffer comprises a salt at a concentration of about 43 mM toabout 45 mM.

The processing buffer can be made in any suitable buffer solution knownthe skilled artisan. Such buffer solutions include, but are not limitedto, TBS, PBS, BIS-TRIS, BIS-TRIS Propane, HEPES, HEPES Sodium Salt, MES,MES Sodium Salt, MOPS, MOPS Sodium Salt, Sodium Chloride, Ammoniumacetate solution, Ammonium formate solution, Ammonium phosphatemonobasic solution, Ammonium tartrate dibasic solution, BICINE bufferSolution, Bicarbonate buffer solution, Citrate Concentrated Solution,Formic acid solution, Imidazole buffer Solution, MES solution, Magnesiumacetate solution, Magnesium formate solution, Potassium acetatesolution, Potassium acetate solution, Potassium acetate solution,Potassium citrate tribasic solution, Potassium formate solution,Potassium phosphate dibasic solution, Potassium phosphate dibasicsolution, Potassium sodium tartrate solution, Propionic acid solution,STE buffer solution, STET buffer solution, Sodium acetate solution,Sodium formate solution, Sodium phosphate dibasic solution, Sodiumphosphate monobasic solution, Sodium tartrate dibasic solution, TNTbuffer solution, TRIS Glycine buffer solution, TRIS acetate-EDTA buffersolution, Triethylammonium phosphate solution, Trimethylammonium acetatesolution, Trimethylammonium phosphate solution, Tris-EDTA buffersolution, TRIZMA® Base, and TRIZMA® HCL. Alternatively, the processingbuffer can be made in water.

In some embodiments, the processing buffer comprises a mixture ofTrirton-X, DNAse I, human plasmin, CaCl₂ and Tween-20. In oneembodiment, the processing buffer consists of a mixture of Trirton-X,DNAse I, human plasmin, CaCl₂ and Tween-20 in a TBS buffer.

In one embodiment, one ml of the processing buffer comprises 100 μl ofTRITON X100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), 10 μl ofDNAse (1 U/1 μl ), 10 μl of human plasmin at 4.6mg/ml and 870 μl of amixture of TBS, 0.1% Tween-20 and 50 mM CaCl₂.

Reagents and treatments for processing blood before assaying are alsowell known in the art, e.g., as used for assays on Abbott TDx, AxSYM®,and ARCHITECT® analyzers (Abbott Laboratories), as described in theliterature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal AntibodyAssay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem.36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYMCyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMITCyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or ascommercially available. Additionally, pretreatment can be done asdescribed in Abbott's U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0471 293, and U.S. Pat. App. Pub. No. 2008/0020401, content of all ofwhich is incorporated herein by reference. It is to be understood thatone or more of these known reagents and/or treatments can be used inaddition to or alternatively to the sample treatment described herein.

In some embodiments, after addition of the processing buffer, the samplecomprises 1% TRITON X(2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), 10 U of DNase, 4.6mg/ml of plasmin, 5 mM Calcium, 0.01% of Tween 20, 2.5 mM of Tris, 150mM of NaCl and 0.2 mM of KCl in addition to the components alreadypresent in the sample.

After addition of the processing buffer, the sample can undergo mixing.This can be simply accomplished by agitating the sample, e.g., shakingor vortexing the sample and/or moving the sample around, if it is in amicrofluidic device. In some embodiments where the microbe-targetingarticle is in the form of a dipstick or a membrane, themicrobe-targeting dipstick or membrane can be dipped in a volume of atest sample and gently agitated with a rocking motion.

After addition of the processing reagents, the sample can be incubatedfor a period of time, e.g., for at least one minute, at least twominutes, at least three minutes, at least four minutes, at least fiveminutes, at least ten minutes, at least fifteen minutes, at least thirtyminutes, at least forty-five minutes, or at least one hour. Suchincubation can be at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature.

1206 (1208 (microbe capture) and 1210 (microbe separation)): Afterprocessing of the sample, the sample can be subjected to a microbecapture process. During the microbe capture process, a microbe-targetingarticle added into a test sample can capture one or more microbespresent in the test sample. In some embodiments, the microbe captureprocess can be repeated and/or performed for a sufficient amount of timeto allow for concentrating and/or cleaning up the test sample beforemicrobe detection. Thus, microbe capture and separation processdescribed herein can be used for concentrating and/or cleaning up asample before analysis for a target component in the sample.

In some embodiments, the microbe capture process can comprise mixing amicrobe-targeting article with the test sample. In some embodiments, themicrobe-targeting article can be already present in the processingbuffer. Amount of the microbe-targeting article added to the sample canbe dependent on a number of different factors, such as, number ofmicrobe-targeting molecules on each article, size of the article, shapeof the article, binding affinity of the microbe-targeting molecule tothe microbe, and concentration of the microbe in the sample.Additionally, amount of the microbe-targeting articles in the sample canbe adjusted to optimize the capture of microbes. In some embodiments,amount of microbe-targeting articles in the sample is such that onemicrobe-targeting article binds with one microbe. However, each microbecan be bound to more than one microbe-targeting article. This can reducecross-linking of multiple microbes together which can lead tocoagulation and/or precipitation of such cross-linked microbes from thesample.

Generally, the total amount of the microbe-binding molecules contactedwith the test sample can range from about 0.01 μg to about 1 mg, about0.1 μg to about 500 μg, about 0.5 μg to about 250 μg, about 1 μg toabout 100 μg, or about 3 μg to about 60 μg. In some embodiments, thetotal amount of the microbe-binding molecules contacted with the testsample can range from about 500 μg to about 1000 mg, about 1 mg to about750 mg, about 5 mg to about 500 mg, about 10 mg to about 250 mg, orabout 25 mg to about 100 mg.

In some embodiments, a plurality of microbe-targeting articles can becontacted with a test sample. The plurality of microbe-targetingarticles can comprise at least two subsets (e.g., 2, 3, 4, 5, or moresubsets), wherein each subset of microbe-targeting articles have apre-determined dimension. In some embodiments, the plurality ofmicrobe-targeting articles can comprise a first subset of themicrobe-targeting articles and a second subset of microbe-targetingarticles. In such embodiments, the first subset of the microbe-targetingarticles each has a first pre-determined dimension; and the secondsubset of the microbe-targeting articles each has a secondpre-determined dimension. Additionally, each subset of themicrobe-targeting articles can comprise on their surfaces substantiallythe same density or different densities of the microbe-binding moleculesdescribed herein.

Different subsets of the plurality of the microbe-targeting articles canbe brought into contact with a test sample in any manner. For example,in some embodiments, the plurality of the microbe-targeting articles canbe provided as a single mixture comprising at least two subsets of themicrobe-targeting articles to be added into a test sample. In someembodiments, in order to distinguish among different subsets of themicrobe-targeting articles, the microbe-targeting articles in eachsubset can have a distinct detection label. For example, themicrobe-targeting articles in each subset can have adistinct-fluorescent label that can be sorted afterward, for example, byflow cytometry.

In other embodiments, the plurality of the microbe-targeting articlescan be brought into contact with a test sample in a sequential manner.For example, a test sample can be contacted with a first subset of themicrobe-targeting articles, followed by a contact with at least one moresubsets of the microbe-targeting articles. The previous subset of themicrobe-targeting articles can be removed from the test sample beforeaddition of another subset of the microbe-targeting articles into thetest sample.

By way of example only, when the microbe-targeting article is amicrobe-targeting molecule coated microparticle (also referred to as acoated-microparticle), generally, about 100 to about 10⁹ microparticlescan be contacted with each ml of the sample. In some embodiments, about10⁴ to about 5×10⁶ coated-microparticless can be contacted with each mlof sample.

As discussed above, in some embodiments, a plurality ofmicrobe-targeting articles can be contacted with a test sample.Accordingly, in some embodiments, a plurality of coated-microparticlescan be contacted with a test sample. The plurality ofcoated-microparticles can comprise at least two subsets (e.g., 2, 3, 4,5, or more subsets), wherein each subset of coated-microparticles have apre-determined dimension. In some embodiments, the plurality ofcoated-microparticles can comprise a first subset of thecoated-microparticles and a second subset of the coated-microparticles.In such embodiments, the first subset of the coated-microparticles eachhas a first pre-determined dimension; and the second subset of thecoated-microparticles each has a second pre-determined dimension. Thepre-determined dimension of a coated-microparticle depends, in part, onthe dimension of a microparticle described herein to which theengineered microbe-binding molecules are conjugated. For example, insome embodiments, the microparticle can have a size of about 10 nm to 10μm, about 20 nm to about 5 μm, about 40 nm to about 1 μm, about 50 nm toabout 500 nm, or about 50 nm to about 200 nm. Additionally, each subsetof the coated-microparticles can comprise on their surfacessubstantially the same density or different densities of themicrobe-targeting molecules disclosed herein.

Different subsets of the plurality of the coated-microbeads can bebrought into contact with a test sample in any manner. For example, insome embodiments, the plurality of the coated-microbeads can be providedas a single mixture comprising at least two subsets of thecoated-microbeads to be added into a test sample. In some embodiments,in order to distinguish among different subsets of thecoated-microbeads, the coated-microbeads in each subset can have adistinct detection label, e.g., a distinctly-fluorescent label that canbe sorted afterward, for example, by flow cytometry.

In some embodiments, the coated-microparticles can be present in theprocessing buffer. In one embodiment, one ml of the processing buffercomprises 100 μl of TRITON X100(2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), 10 μl of a solutioncomprising about 25 million coated-microparticles, 10 μl of DNAse (1 U/1μl), 10 μl of human plasmin at 4.6 mg/ml and 870 μl of a mixture of TBS,0.1% Tween-20. In some embodiments, the processing buffer can include acalcium salt, e.g., CaCl₂ (e.g., ˜50 mM CaCl₂). In some embodiments, theprocessing or capture buffer can include no calcium salt, e.g., CaCl₂.

After addition of the microbe-targeting articles, the microbe-targetingarticles can be mixed in the sample to allow microbes to bind with themicrobe-targeting molecules. This can be simply accomplished byagitating the sample, e.g., shaking or vortexing the sample and/ormoving the sample around in a microfluidic device. In some embodimentswhere the microbe-targeting article is in a form of a dipstick or amembrane, the microbe-targeting dipstick or membrane can be dipped in avolume of a test sample and gently agitated with a rocking motion.

The volume of the test sample required for contacting themicrobe-targeting article can vary with, e.g., the selection of themicrobe-targeting article (e.g., microbeads, fibers, filters, filters,fibers, screens, mesh, tubes, hollow fibers), the concentration ofmicrobes present in the test sample, the platform used to carry out theassay (e.g., a microfluidic device, a blood collection tube, amicrotiter plate, or like). For example, if the assay is performed in amicrofluidic device, the test sample volume used to perform the assaycan range from about 1 μL to about 500 μL, from about 5 μL to about 250μL, or from about 10 μL to about 100 μL. In some embodiments, if theassay is performed in a test tube, the test sample volume can range fromabout 0.05 mL to about 50 mL, from about 0.25 ml to about 50 ml, about0.5 ml to about 25 ml, about 1 ml to about 15 ml, or about 2 ml to about10 ml. In some embodiments, the test sample volume used to perform theassay described herein can be about 1 mL to about 5 ml. In oneembodiment, the test sample volume used to perform the assay describedherein is about 5 ml to about 10 mL.

After contacting the test sample with the microbe-targeting molecules(e.g., with a microbe-targeting article), the sample mixture can beincubated for a period of time to allow the microbe of interest to bindonto the microbe-targeting molecules on the microbe-targeting article.Such incubation can be fr any desired period of time to allow sufficientnumber of microbes to bind to the microbe-targeting molecules. Forexample, the incubation can be for at least one minute, at least twominutes, at least three minutes, at least four minutes, at least fiveminutes, at least ten minutes, at least fifteen minutes, at least abouttwenty minutes, at least thirty minutes, at least forty-five minutes, orat least one hour. In one embodiment, the sample mixture can beincubated for a period of about 10-20 minutes. Further, such incubationcan be performed at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the incubation can be performed at atemperature ranging from about room temperature to about 37° C. In someembodiments, the sample can be incubated for about 10 mins to about 20mins at room temperature. In some embodiments, the sample is incubatedfor about fifteen minutes at room temperature.

To prevent or reduce agglutination (or non-specific binding) duringseparation of the microbes from the sample, additional reagents can beadded to the sample mixture. Such reagents are also referred to asblocking reagents herein. For example, these blocking reagents cancomprise a ligand of the affinity molecules on the coated-microbeads.Addition of such blocking reagents can reduce agglutination by bindingwith any empty ligand binding sites on the affinity molecules.Accordingly, when microbe-targeting magnetic microbeads are used forcapturing the microbes, the blocking reagent can be a carbohydrate, suchas mannose. Amount of additional reagent can depend on the amount ofmicrobeads added to the sample. Generally, about the reagent is added toa final concentration of about 0.1 mM to about 10 mM. The amount of theblocking agent required can vary, at least partly, with the amountand/or surface area of the microbe-targeting substrate that is incontact with a test sample. In some embodiments, the blocking reagentcan be added to a final concentration of about 0.1% (w/v) to about 10%(w/v), about 0.5% (w/v) to about 7.5% (w/v), or about 1% (w/v) to about5% (w/v). In some embodiments, about 1% casein can be used as a blockingagent in the assay described herein.

After addition of the blocking reagent, the sample mixture can beincubated for a period of time to allow the blocking reagent to bind towith the microbe-targeting molecules, e.g., for at least one minute, atleast two minutes, at least three minutes, at least four minutes, atleast five minutes, at least ten minutes, at least fifteen minutes, atleast thirty minutes, at least forty-five minutes, or at least one hour.Such incubation can be at any appropriate temperature, e.g.,room-temperature (e.g., about 16° C. to about 30° C.), a coldtemperature (e.g. about 0° C. to about 16° C.), or an elevatedtemperature (e.g., about 30° C. to about 95° C.). In some embodiments,the sample is incubated for about fifteen minutes at room temperature.In some embodiments, incubation is for about 5 seconds to about 60seconds. In some embodiments, the incubation can be performed at atemperature ranging from about room temperature to about 37° C. In someembodiments, the sample is incubated for about fifteen minutes at roomtemperature.

To prevent or reduce non-specific binding during the contact between amicrobe-targeting substrate and a test sample, in some embodiments, themicrobe-targeting article (e.g., coated-microparticles) or the testsample can be pre-treated with a blocking agent that does not react withmicrobes, before contacting each other. Exemplary blocking agentsinclude, but are not limited t, casein, normal serum, BSA, non-fat drymilk powder and any art-recognized block agent. Optionally,microbe-targeting article after blocking can be washed with anyart-recognized buffer to remove any leftover blocking agent. The numberof wash steps can range from 1 to many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more wash steps. In one embodiment, the microbe-targetingsubstrate after blocking can be washed with a buffer, e.g., TBST, forabout at least 1-3 times.

After incubation, the microbe-targeting article can then be analyzed, asdescribed below, for the presence or absence of a bound microbe. In someembodiments, the test sample can be subjected to a second microbecapturing/detection assay. The second microbe capturing/detection assaycan be carried out using a second microbe-targeting molecule which doesnot comprises CRP or a fragment thereof. For example, the secondmicrobe-targeting molecule comprises at least one first domaincomprising at least a portion of a microbe-binding domain of amicrobe-binding protein, wherein the microbe-binding protein is not aCRP; a second domain, as described in this disclosure; and a linkerconjugating the first and the second domains. Microbe-binding domainsthat do not comprise CRP are described elsewhere in the disclosure.Exemplary second microbe-targeting molecules are described, for example,in PCT Application No. PCT/US2011/021603 filed Jan. 19, 2011 and No.PCT/US2012/047201, filed Jul. 18, 2012, and U.S. Provisional ApplicationNo. 61/691,983 filed Aug. 22, 2012, contents of all of which areincorporated herein by reference in their entireties. In someembodiments, the second microbe-targeting molecule comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 9,11, 12, and 23-27.

Exemplary optional modifications to 1208 (Microbe capture): Inaccordance with one aspect described herein, the test sample can becontacted with a microbe-targeting molecule in the presence of achelating agent. Without wishing to be bound by theory, the addition ofa chelating agent to a test sample and/or processing buffer can reducethe likelihood of any protein A- and protein G-negative microbe, but notprotein A- or protein G-expressing microbe in the test sample, to bindwith at least one microbe-binding molecule. Accordingly, detection ofany microbes bound on the microbe-targeting substrate described hereinin the presence of a chelating agent can determine the presence orabsence of a protein A- or protein G-expressing microbe in a testsample.

The chelating agent can be added into the processing buffer comprisingthe test sample. The amount of the chelating agent is sufficient tochelate free calcium ions and thus prevent or reduce calcium-dependentcarbohydrate recognition domain binding (e.g., mannose-binding lectin)with a microbe. The amount of the chelating agent needed to prevent orreduce calcium-dependent carbohydrate recognition domain binding (e.g.,mannose-binding lectin) with a microbe can depend on, e.g., theconcentration of free calcium ions present in a test sample andoptionally a capture buffer, e.g., used to dilute a chelating agentand/or a test sample. Thus, in some embodiments, the concentration ofthe chelating agent can be higher than the total concentration of freecalcium ions present in the combined solution of a test sample and acapture buffer. For example, in some embodiments, the concentration ofthe chelating agent can be at least about 30% higher, including at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, up to and including 100%, or any percent between about 30%and about 100%, higher than the total concentration of free calcium ionspresent in the combined solution of a test sample and a capture buffer.In other embodiments, the concentration of the chelating agent can be atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 6-fold, atleast about 7-fold, at least about 8-fold, at least about 9-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 30-fold, at least about 40-fold, at least about 50-fold, atleast about 75-fold, at least about 100-fold or more, higher than thetotal concentration of free calcium ions present in the combinedsolution of a test sample and a capture buffer. In one embodiment, theconcentration of the chelating agent can be at least about 5-fold toabout 50-fold, or at least about 7-fold to about 25-fold, higher thanthe total concentration of free calcium ions present in the combinedsolution of a test sample and a capture buffer.

In some embodiments, the concentration of a chelating agent present inthe test sample and optionally a processing or capture buffer, e.g.,used to dilute the chelating agent or the test sample, can range fromabout 0.1 mM to about 1 M, about 10 mM to about 500 mM, about 20 mM toabout 250 mM, or about 25 mM to about 125 mM. In one embodiment, theconcentration of a chelating agent present in the test sample andoptionally a capture buffer can be about 25 mM to about 125 mM.

In some embodiments, the concentration of a chelating agent present inthe test sample containing the microbe-targeting substrate can besufficient to reduce the likelihood of a protein A- and proteinG-negative microbe, if present in the test sample, to bind with at leastone microbe-binding molecule. For example, the concentration of achelating agent present in the test sample with the microbe-targetingsubstrate can be sufficient to reduce the number of protein A- andprotein G-negative microbes, if present in the test sample, to bind withat least one microbe-binding molecule, by at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least 80% or higher, as compared to the number of protein A- andprotein G-negative microbes bound on the microbe binding molecules inthe absence of the chelating agent. In some embodiments, theconcentration of a chelating agent present in the test sample with themicrobe-targeting substrate can be sufficient to reduce the number ofprotein A- and protein G-negative microbes, if present in the testsample, to bind with at least one microbe-binding molecule, by at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, up to and including 100%, or any values betweenabout 85% and about 100%, as compared to the number of protein A- andprotein G-negative microbes bound on the microbe-binding molecules inthe absence of the chelating agent.

The protein A-expressing and protein G-expressing microbes can generallybind to microbe-binding molecules via two independent (but additive)mechanisms: Fc-mediated binding and microbe surface-binding domainmediated binding. Without wishing to be bound by theory, while theprotein A-expressing and protein G-expressing microbes can still becaptured on the microbe-targeting molecules in the presence of achelating agent, the presence of free calcium ions can further increasethe number of protein A-expressing and protein G-expressing microbesbound to the microbe-targeting molecules, because the overall binding inthe presence of calcium ions can be almost twice as strong as in theabsence of calcium ions.

Accordingly, in some embodiments, the concentration of a chelating agentpresent in the test sample containing the microbe-targeting articles canreduce the number of protein A-expressing microbes or proteinG-expressing microbes bound onto the microbe-targeting substrate, butsuch effect as compared to that on the protein A- and protein G-negativemicrobes is much smaller, e.g., at least about 30% smaller, at leastabout 40% smaller, at least about 50%, at least about 60% smaller, atleast about 70% smaller, or at least about 80% smaller.

In some embodiments, the concentration of a chelating agent used in theassay described herein can be high enough to prevent at least about 80%or higher, including at least about 90%, at least about 95%, up to andincluding 100%, of the protein A- and protein G-negative microbes frombinding to be microbe-targeting substrate, but low enough to allow atleast about 30% or higher, including at least about 40%, at least about50%, at least about 60%, at least about 70% or higher, of the proteinA-expressing microbes or protein G-expressing microbes to bind with themicrobe-targeting substrate. In one embodiment, the concentration of achelating agent used in the assay described herein can be high enough toprevent at least about 90% or higher, of the protein A- and proteinG-negative microbes, if any present in the test sample, from binding tobe microbe-targeting substrate, but low enough to allow at least about50% of the protein A-expressing microbes or protein G-expressingmicrobes, if any present in the test sample, to bind with themicrobe-targeting substrate.

Examples of calcium ion-chelating agents can include, but are notlimited to, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid,ethylenediaminetetraacetic acid (EDTA); ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), abuffer containing citrate,N,N-Bis(2-(bis-(carboxymethyl)amino)ethyl)-glycine (DTPA),nitrilo-2,2′,2″-triacetic acid (NTA), a buffer that precipitates acalcium ion from the test sample, including, e.g., a phosphate buffer, acarbonate buffer and a bicarbonate buffer, a low pH buffer (e.g., a pHbuffer less than pH 7 or less than pH 6), citric acids and its salts,gluconic acid and its salts, alkali metal pyrophosphates, alkali metalpolyphosphates, sodium hexametaphosphate, triethylene tetramine,diethylene triamine, o-phenanthroline, oxalic acid and any combinationsthereof.

The chelating agent can be directly added to the test sample or preparedin a processing or capture buffer, which is then added to the testsample in contact with the microbe-targeting substrate. The processingor capture buffer can be any buffered solutions, e.g., with a pH rangingfrom about 6 to about 10. In some embodiments, the processing or capturebuffer can include, but is not limited to, a tris-buffered saline, aphosphate buffered saline or a combination thereof. In some embodiments,the processing or capture buffer can include a surfactant, e.g., toprevent non-specific binding of a microbe to a microbe-surface-bindingdomain of the microbe-targeting substrate, and/or to saturatenon-specific binding sites, if any, present in the microbe-targetingsubstrate. A surfactant or detergent, e.g., as described earlier, can bedissolved in a buffered solution in any amount, e.g., ranging from about0.001% (v/v) to about 5% (v/v), from about 0.01% (v/v) to about 2.5%(v/v), or from about 0.05% (v/v) to about 1% (v/v). In some embodiments,the surfactant added to the processing or capture buffer can includeTween 80 or polysorbate 80 at a concentration of about 0.01% to about0.1%. In one embodiment, the surfactant added to the processing orcapture buffer can include Tween 80 or polysorbates 80 at aconcentration of about 0.05%.

After incubation, the microbe-targeting article can then be analyzed, asdescribed below, for the presence or absence of a bound microbe. In theabsence of a microbe-targeting article-bound microbe, in someembodiments, the previous volume of the test sample or a new freshvolume of the test sample can be contacted with a freshmicrobe-targeting substrate in the presence of free calcium ions, e.g.,to determine the presence or absence of protein A- and proteinG-negative microbes. In some embodiments, the free calcium ions can beproduced adding a sufficient amount of calcium salts in the test sample.If there has been a chelating agent present in the test sample, a higheramount of calcium salts is generally needed in order to obtain freecalcium ions.

As used herein, the term “free calcium ions” refers to calcium ions thatare not complexed with any molecule or compound, e.g., a chelatingagent, which can hinder its reaction with other molecules or ions tomediate binding of carbohydrate patterns on a microbial cell surface toa microbe surface-binding domain (e.g., MBL) of the engineeredmicrobe-binding molecule. Accordingly, in some embodiments, free calciumions can be present in the absence of chelating agent. In someembodiments, free calcium ions can be present in a solution comprising achelating agent and calcium ions. In some embodiments, the amount ofcalcium ions present in the solution is at least about 30% more than anamount sufficient to interact with substantially all the chelating agentmolecules present in the solution to form chelate complexes. Forexample, in some embodiments, in order to obtain free calcium ions, theamount of calcium ions present in the solution can be at least about30%, including at least about 40%, at least about 50% , at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 98%, up to and including 100% and anypercent between 30% and 100%, more than an amount sufficient to interactwith substantially all the chelating agent molecules present in thesolution to form chelate complexes. In some embodiments, in order toobtain free calcium ions, the amount of calcium ions present in thesolution can be at least about 1-fold, at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, at leastabout 6-fold, at least about 7-fold, at least about 8-fold, at leastabout 9-fold, at least about 10-fold, at least about 15-fold, at leastabout 20-fold, at least about 25-fold, at least about 50-fold, at leastabout 100-fold, at least about 500-fold, at least about 1000-fold, morethan an amount sufficient to interact with substantially all thechelating agent molecules present in the solution to form chelatecomplexes. In some embodiments, free calcium ions can be present in asolution when the concentration of calcium ions in the solution is atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 6-fold, atleast about 7-fold, at least about 8-fold, at least about 9-fold, atleast about 10-fold, at least about 20-fold, or higher than theconcentration of a chelating agent present in the same solution.

In some embodiments, calcium ions can be obtained from a water-solublecalcium salt. By the term “water-soluble calcium salt” is meant acalcium salt which has significant solubility in water at roomtemperature, for example at least 1 gram per 100 ml water, at least 10grams per 100 ml water, or at least 25 grams per 100 ml water or higher.Examples of calcium salts include, without limitations, calciumchloride, calcium fluoride, calcium bromide, calcium iodide, calciumnitrate, calcium citrate, calcium formate, calcium acetate, calciumgluconate, calcium ascorbate, calcium lactate, calcium glycinate andmixtures thereof. In some embodiments, calcium chloride can be used as asource of calcium ions.

Free calcium ions can be present at a concentration or an amountsufficient to mediate binding of calcium-dependent carbohydraterecognition domain with a microbe surface. In some embodiments, freecalcium ions can be present at a concentration of at least about 1 μM,at least about 10 μM, at least about 25 μM, at least about 50 μM, atleast about 100 μM, at least about 250 μM, at least about 500 μM, or atleast about 1 mM or higher. In some embodiments, the free calcium ionscan be present at a concentration of at least about 1 mM, at least about2.5 mM, at least about 5 mM, at least about 10 mM, at least about 25 mM,at least about 50 mM, at least about 75 mM, at least about 100 mM orhigher. In other embodiments, the free calcium ions can be present at aconcentration of at least about 100 mM, at least about 150 mM, at leastabout 200 mM, at least about 300 mM, at least about 400 mM, at leastabout 500 mM, at least about 600 mM, at least about 700 mM, at leastabout 800 mM, at least about 900 mM, at least about 1 M or higher. Inone embodiment, the free calcium ions can be present at a concentrationof about 1 mM to about 10 mM. In one embodiment, the free calcium ionscan be present at a concentration of at least about 5 mM.

While a chelating agent can be added during an initial capture of amicrobe on a microbe-targeting substrate, the chelating agent can alsobe first excluded to allow the initial capture of any microbe, includingprotein A- and protein G-negative microbes, on a microbe-targetingsubstrate in the presence of free calcium ions, but added after thecapture to remove any captured protein A- or protein G-negative microbesfrom the microbe-targeting substrate.

Accordingly, in some embodiments, the microbe capture can comprise (i)contacting at least a first volume of a test sample with amicrobe-targeting substrate described herein in the presence of freecalcium ions, and (ii) contacting the microbe-binding molecule of themicrobe-targeting substrate described herein, upon the contact with thetest sample, with a solution comprising a chelating agent.

When the microbe-targeting substrate is contacted with a test sample inthe presence of free calcium ions as described herein, microbes thatprimarily depend on calcium-dependent MBL-mediated binding such asprotein A- and protein G-negative microbes, e.g., E. coli can bind tothe microbe-target substrate, in addition to microbes associated withFc-mediated binding such as protein A-expressing microbes (e.g., S.aureus), and protein G-expressing microbes.

To elute off or remove from the microbe-targeting substrate the capturedmicrobes that primarily depend on calcium-dependent MBL-mediated bindingsuch as protein A- and protein G-negative microbes, e.g., E. coli, themicrobe-binding molecules on the microbe-targeting substrates can becontacted with a solution comprising a sufficient amount of a chelatingagent as described herein. The solution comprising the chelating agentcan be same as a capture buffer described above. In such embodiments,the microbe-targeting substrate can be incubated with the solutioncomprising a chelating agent for a period of time to allow microbes thatprimarily bind to microbe-binding molecules via calcium-dependentMBL-mediated binding to elute off the microbe-targeting substrate, e.g.,incubation for at least one minute, at least two minutes, at least threeminutes, at least four minutes, at least five minutes, at least tenminutes, at least fifteen minutes, at least thirty minutes, at leastforty-five minutes, or at least one hour. Such incubation can beperformed at any appropriate temperature, e.g., room-temperature (e.g.,about 16° C. to about 30° C.), a cold temperature (e.g. about 0° C. toabout 16° C.), or an elevated temperature (e.g., about 30° C. to about95° C.). In some embodiments, the microbe-targeting substrate can beincubated with the solution comprising a chelating agent for at leastabout 5 mins to about 15 mins at room temperature.

In these embodiments, the concentration of a chelating agent used in theassay described herein is sufficient to elute off or remove from themicrobe-targeting substrate at least about 30% of the bound protein A-and protein G-negative microbes (e.g., E. coli). For example, theconcentration of a chelating agent used in the assay described herein issufficient to elute off or remove from the microbe-targeting substrateat least about 30% of the bound protein A- and protein G-negativemicrobes (e.g., E. coli), including at least about 40%, at least about50%, at least about 60%, at least about 70%, at least 80% or higher, ofthe bound protein A- and protein G-negative microbes (e.g., E. coli). Insome embodiments, the concentration of a chelating agent used in theassay described herein is sufficient to elute off or remove from themicrobe-targeting substrate at least about 85% of the bound protein A-and protein G-negative microbes (e.g., E. coli), including at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,up to and including 100%, or any values between about 85% and about100%, of the bound protein A- and protein G-negative microbes (e.g., E.coli).

As noted above, the protein A-expressing and protein G-expressingmicrobes can bind to microbe-binding molecules via Fc-mediated andcalcium ion-dependent MBL-mediated binding. Without wishing to be boundby theory, the concentration of a chelating agent used in the assaydescribed herein can also elute off or remove at least a portion of theprotein A-expressing and/or protein G-expressing microbes from themicrobe-targeting substrate. For example, the concentration of achelating agent used to elute off or remove protein A- and proteinG-negative microbes from the microbe-targeting substrate can besufficient to elute off or remove no more than 60%, no more than 50%, nomore than 40%, no more than 30%, no more than 20%, no more than 10% orlower, of the bound protein A-expressing or protein G-expressingmicrobes. In some embodiments, the concentration of a chelating agentused to elute off or remove from the microbe-targeting substrate atleast about 80% or more, including at least about 90% or more, of thebound protein A- and protein G-negative microbes can be sufficient toelute off or remove no more than 50%, or more than 40% of the boundprotein A-expressing and/or protein G-expressing microbes.

As a person having ordinary skill in the art can appreciate, the assaydescribed herein can further comprise isolating the microbe-targetingsubstrate from the test sample, e.g., as described below, beforecontacting microbe-binding molecules on its substrate surface with thesolution comprising the chelating agent described herein.

1210 (Microbe separation from sample): The sample mixture can be thensubjected to a microbe separation process. In some embodiments, becausemicrobes are bound with one or more magnetic microparticles, a magnetcan be employed to separate the bound microbes from the test sample. Theskilled artisan is well aware of methods for carrying out magneticseparations. Generally, a magnetic field gradient can be applied todirect the capture of magnetic microbeads. Optionally, the bound microbecan be washed with a buffer to remove any leftover sample and unboundcomponents. Number of wash steps can range from 1 to many, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. Without wishing to be boundby a theory, capture and separation of the bound microbes from thesample can concentrate the microbes and also remove components, whichcan interfere with the assay or process, from the test sample.

The magnetic field source can be any magnet device positioned togenerate the magnetic field gradient that is used to pull the capturedmicrobe out from the sample. An electromagnetic controller can be usedto control and adjust the magnetic field and gradients thereof, and tocontrol the migration, separation and orientation of the magneticallybound microbes. The magnetic field gradient can be generated by apermanent magnet or by an electromagnetic signal generator. Theelectromagnetic signal generator can include an electromagnet orelectrically-polarizable element, or at least one permanent magnet. Themagnetic field gradient can be produced at least in part according to apre-programmed pattern. The magnetic field gradient can have a definedmagnetic field strength and/or spatial orientation. In some embodiments,the magnetic field gradient has a defined magnetic field strength. Theterm “magnetic field gradient” as used herein refers to a variation inthe magnetic field with respect to position. By way of example only, aone-dimensional magnetic field gradient is a variation in the magneticfield with respect to one direction, while a two-dimensional magneticfield gradient is a variation in the magnetic field with respect to twodirections.

As used herein, the term “magnetic field” refers to magnetic influenceswhich create a local magnetic flux that flows through a composition andcan refer to field amplitude, squared-amplitude, or time-averagedsquared-amplitude. It is to be understood that magnetic field can be adirect-current (DC) magnetic field or alternating-current (AC) magneticfield. The magnetic field strength can range from about 0.00001 Teslaper meter (T/m) to about 10⁵ T/m. In some embodiments, the magneticfield strength can range from about 0.0001 T/m to about 10⁴ T/m. In someother embodiments, the magnetic field strength can range from about0.001 T/m to about 10³ T/m.

In some embodiments, microbe capture and/or microbe-targeting substrateseparation can be performed by a rapid microbe diagnostic assay ordevice as described in Int. Pat. App. No. WO 2011/091037, filed Jan. 19,2011, the content of which is incorporated herein by reference. A rapidmicrobe diagnostic device as described in Int. Pat. App. No. WO2011/091037, filed Jan. 19, 2011, can be modified to replace the capturechamber or capture and visualization chamber with an s-shaped flow path.A magnet can then be used to capture bound microbe against the flow pathwall; separating the bound microbe from rest of the sample.

In some embodiments, microbe capture and/or separation is by a device ormethod as described in U.S. Pat. App. Pub. No. 2009/0220932, No.2009/007861, No. 2010/0044232, No. 2007/0184463, No. 2004/0018611, No.2008/0056949, No. 2008/0014576, No. 2007/0031819, No. 2008/0108120, andNo. 2010/0323342, the contents of which are all incorporated herein byreference.

In some embodiments, microbe capture, separation, or detection is by adevice or method as described in PCT Application No. PCT/US2013/028409,filed Feb. 28, 2013, No. PCT/US2012/031864, filed Feb. 4, 2012, and No.PCT/US2011/021718 filed Jan. 19, 2011; U.S. patent application Ser. No.13/918,193 filed Jun. 14, 2013; and U.S. Prov. App. No. No. 61/788,570filed Mar. 15, 2013, No. 61/772,436 filed Mar. 4, 2013, No. 61/772,360filed Mar. 4, 2013, and No. 61/673,071 filed Jul. 18, 2013, the contentsof which are all incorporated herein by reference.

Without limitations, if a microbe-targeting substrate does not possess amagnetic property, isolation of a microbe-targeting substrate (e.g.,particles, posts, fibers, dipsticks, membrane, filters, capillary tubes,etc.) from the test sample can be carried out by non-magnetic means,e.g., centrifugation, and filtration. In some embodiments where themicrobe-targeting substrate is in a form a dipstick or membrane, themicrobe-targeting dipstick or membrane can be simply removed from thetest sample, where microbes, if any, in the test sample, remained boundto the engineered microbe-binding molecules conjugated to the dipstickor membrane substrate.

Optionally, the microbe-targeting substrate after isolated from the testsample or processing buffer can be washed with a buffer (e.g., TBST) toremove any residues of test sample, solution comprising the chelatingagent or any unbound microbes. The number of wash steps can range from 1to many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. In oneembodiments, the microbe-targeting substrate after isolated from thesolution comprising the chelating agent and/or the test sample can bewashed with a buffer (e.g., TBST) for about at least 1-3 times.

1212 (Microbe detection/analysis): A detection component, device orsystem can be used to detect and/or analyze the presence of theseparated microbe, for example, by spectroscopy, electrochemicaldetection, polynucleotide detection, fluorescence anisotropy,fluorescence resonance energy transfer, electron transfer, enzyme assay,magnetism, electrical conductivity, isoelectric focusing,chromatography, immunoprecipitation, immunoseparation, aptamer binding,filtration, electrophoresis, use of a CCD camera, immunoassay, ELISA,Gram staining, immunostaining, microscopy, immunofluorescence, westernblot, polymerase chain reaction (PCR), RT-PCR, fluorescence in situhybridization, sequencing, mass spectroscopy, or substantially anycombination thereof. The separated microbe can remain bound on themicrobe-targeting substrate during detection and/or analysis, or beisolated form the microbe-targeting substrate prior to detection and/oranalysis.

In some embodiments, labeling molecules that can bind with the microbecan also be used to label the microbes for detection. As used herein, a“labeling molecule” refers to a molecule that comprises a detectablelabel and can bind with a target microbe. Labeling molecules caninclude, but are not limited to, an engineered microbe-targetingmolecule disclosed herein, wheat germ agglutinin, lectins, antibodies(e.g., gram-negative antibodies or gram-positive antibodies, antibioticsto specific microbial strains or species), antigen binding fragments ofantibodies, aptamers, ligands (agonists or antagonists) of cell-surfacereceptors and the like. The labeling molecule can also be a non-specificlabeling molecule that non-specifically stains all viable cells in asample.

Any method known in the art for detecting the particular label can beused for detection. Exemplary methods include, but are not limited to,spectrometry, fluorometry, microscopy imaging, immunoassay, and thelike. While the microbe capture step can specifically capture microbes,it can be beneficial to use a labeling molecule that can enhance thisspecificity. If imaging, e.g., microscopic imaging, is to be used fordetecting the label, the staining can be done either prior to or afterthe microbes have been laid out for microscopic imaging. Additionally,imaging analysis can be performed via automated image acquisition andanalysis.

For optical detection, including fluorescent detection, more than onestain or dye can be used to enhance the detection or identification ofthe microbe. For example, a first dye or stain can be used that can bindwith a genus of microbes, and a second dye or strain can be used thatcan bind with a specific microbe. Colocalization of the two dyes thenprovides enhanced detection or identification of the microbe by reducingfalse positive detection of microbes.

In some embodiments, microscopic imaging can be used to detect signalsfrom label on the labeling agent. Generally, the microbes in thesubsample are stained with a staining reagent and one or more imagestaken from which an artisan can easily count the number of cells presentin a field of view.

In particular embodiments, microbe can be detected through use of one ormore enzyme assays, e.g., enzyme-linked assay (ELISA). Numerous enzymeassays can be used to provide for detection. Examples of such enzymeassays include, but are not limited to, beta-galactosidase assays,peroxidase assays, catalase assays, alkaline phosphatase assays, and thelike. In some embodiments, enzyme assays can be configured such that anenzyme will catalyze a reaction involving an enzyme substrate thatproduces a fluorescent product. Enzymes and fluorescent enzymesubstrates are known and are commercially available (e.g.,Sigma-Aldrich, St. Louis, Mo.). In some embodiments, enzyme assays canbe configured as binding assays that provide for detection of microbe.For example, in some embodiments, a labeling molecule can be conjugatedwith an enzyme for use in the enzyme assay. An enzyme substrate can thenbe introduced to the one or more immobilized enzymes such that theenzymes are able to catalyze a reaction involving the enzyme substrateto produce a detectable signal.

In some embodiments, an enzyme-linked assay (ELISA) can be used todetect signals from the labeling molecule. In ELISA, the labelingmolecule can comprise an enzyme as the detectable label. Each labelingmolecule can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) enzymes. Additionally, each labeling molecule can comprise oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) sites for bindingwith a microbe. Without wishing to be bound by a theory, presence ofmultimeric probe molecules can enhance ELISA signal.

For ELISA, any labeling molecule conjugated to an enzyme can be used.Exemplary labeling molecule include those comprising the microbe-bindingmolecules comprising detectable labels, wheat germ agglutinin, lectins,antibodies (e.g., gram-negative antibodies or gram-positive antibodies),antigen binding fragments of antibodies, aptamers, ligands (agonists orantagonists) of cell-surface receptors and the like. In someembodiments, the labeling molecule can be a microbe-targeting moleculedisclosed herein, wherein the microbe-targeting molecule comprises adetectable label. In some embodiments, the labeling molecule can be amicrobe-targeting molecule as described, for example, in PCT ApplicationNo. PCT/US2011/021603 filed Jan. 19, 2011 and No. PCT/US2012/047201,filed Jul. 18, 2012, and U.S. Provisional Application No. 61/691,983filed Aug. 22, 2012, contents of all of which are incorporated herein byreference in their entireties.

Similarly, a variety of enzymes can be used, with either colorimetric orfluorogenic substrates. In some embodiments, the reporter-enzymeproduces a calorimetric change which can be measured as light absorptionat a particular wavelength. Exemplary enzymes include, but are notlimited to, beta-galactosidases, peroxidases, catalases, alkalinephosphatases, and the like. In some embodiments, the enzyme is ahorseradish peroxidase (HRP) or an alkaline peroxidase (AP).

A microbe-binding molecule and the detectable label can be linked toeach other by a linker. In some embodiments, the linker between themicrobe-binding molecule and the detectable label an amide bond. In someembodiments, the linker between the microbe-binding molecule and thedetectable label is a disulfide (S—S) bond. When the microbe-bindingmolecule is a peptide, polypeptide or a protein, the detectable labelcan be linked at the N-terminus, the C-terminus, or at an internalposition of the microbe-binding molecule. Similarly, when the detectablelabel is an enzyme, the enzyme can be linked by its N-terminus,C-terminus, or an internal position.

In one embodiment, the ELISA probe molecule can comprise amicrobe-targeting molecule comprising a CRP (or a portion there of)inked to HRP or AP. Conjugation of HRP or AP to peptides, proteins, andantibodies are known in the art. In one embodiment, the construct can begenerated by direct coupling of the enzyme to the microbe-targetingmolecule using a commercially-available conjugation kit.

In some embodiments, the microbes isolated from or remained bound on themicrobe-targeting substrate can be incubated with the enzyme labeledmicrobe-binding molecules for a period of time, e.g., at least about 5mins, at least about 10 mins, at least about 15 mins, at least about 20mins, at least about 25 mins, at least about 30 mins. The typicalconcentrations of enzyme-labeled molecules used in the ELISA assay canrange from about 1:500 to about 1:20,000 dilutions. In one embodiment,the concentration of enzyme-labeled microbe-binding molecules can beabout 1:1000 to about 1:10000 dilutions.

Following incubation with the ELISA probe molecules, the sample can bewashed with a wash buffer one or more (e.g., 1, 2, 3, 4, 5 or more)times to remove any unbound probes. An appropriate substrate for theenzyme (e.g., HRP or AP) can be added to develop the assay. Chromogenicsubstrates for the enzymes (e.g., HRP or AP) are known to one of skillin the art. A skilled artisan can select appropriate chromogenicsubstrates for the enzyme, e.g., TMB substrate for the HRP enzyme, orBCIP/NBT for the AP enzyme. In some embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain calcium ionsat a concentration of about at least about 0.01 mM, at least about 0.05mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM,at least about 2.5 mM, at least about 5 mM, at least about 10 mM, atleast about 20 mM, at least about 30 mM, at least about 40 mM, at leastabout 50 mM or more. In alternative embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain no calciumions. In some embodiments, the wash buffer used after incubation with anELISA probe molecule can contain a chelating agent. A wash buffer can beany art-recognized buffer used for washing between incubations withantibodies and/or labeling molecules. An exemplary wash buffer caninclude, but is not limited to, TBST.

In some embodiments, without wishing to be bound by theory, it can bedesirable to use a wash buffer without a surfactant or a detergent forthe last wash before addition of a chromogenic substrate, because asurfactant or detergent may have adverse effect to the enzymaticreaction with a chromogenic substrate.

One advantage of the ELISA-based approach is that the microbe-targetingarticle does not need to be dispersed or dissociated from the microbebefore binding the secondary reagents. This is in contrast tomicroscopic techniques, in which excess residual solid substrate mayobscure the microbe during imaging. Furthermore, the optical readoutcomponents for ELISA are likely cheaper than in the microscopy case, andthere is no need for focusing or for demanding that the sample be on thesame focal plane. A further advantage of the ELISA-based approach isthat it can take advantage of commercially available laboratoryequipment. In particular, when the solid substrate is magnetic, magneticseparation can be automated using the KINGFISHER® system, the briefculture can be performed using an airlift fermenter, and thecolorimetric/fluorescent readout can be attained using a standard platereader.

Further amplification of the ELISA signal can be obtained bymultimerizing the recognition molecule (e.g., the microbe-bindingmolecule) or by multimerizing the detection enzyme (HRP, etc.). Forinstance, phage expression can be used to yield multimerizedmicrobe-targeting molecules and provide a scaffold to increase theconcentration of enzyme, either through direct coupling of enzyme to thephage particles or using an enzyme conjugated antibody.

In some embodiments, microbe can be detected through use of immunoassay.Numerous types of detection methods may be used in combination withimmunoassay based methods.

In some embodiments, detection of microbes in a sample can also becarried out using light microscopy with phase contrast imaging based onthe characteristic size (5 um diameter), shape (spherical to elliptical)and refractile characteristics of target components such as microbesthat are distinct from all normal blood cells. Greater specificity canbe obtained using optical imaging with fluorescent or cytochemicalstains that are specific for all microbes or specific subclasses (e.g.calcofluor (1 μM to 100 μM) for chitin in fungi, fluorescent antibodiesdirected against fungal surface molecules, gram stains, acid-faststains, fluorescent microbe-targeting molecules, etc.

Microbe detection can also be carried out using an epifluorescentmicroscope to identify the characteristic size (5 um diameter), shape(spherical to elliptical) and staining characteristics of microbes. Forexample, fungi stain differently from all normal blood cells, stronglybinding calcofluor (1 μM to 100 μM) and having a rigid ellipsoid shapenot found in any other normal blood cells.

In some embodiments, a microbe can be detected through use ofspectroscopy. Numerous types of spectroscopic methods can be used.Examples of such methods include, but are not limited to, ultravioletspectroscopy, visible light spectroscopy, infrared spectroscopy, x-rayspectroscopy, fluorescence spectroscopy, mass spectroscopy, plasmonresonance (e.g., Cherif et al., Clinical Chemistry, 52:255-262 (2006)and U.S. Pat. No. 7,030,989; herein incorporated by reference), nuclearmagnetic resonance spectroscopy, Raman spectroscopy, fluorescencequenching, fluorescence resonance energy transfer, intrinsicfluorescence, ligand fluorescence, and the like.

In some embodiments, a microbe can be detected through use offluorescence anisotropy. Fluorescence anisotropy is based on measuringthe steady state polarization of sample fluorescence imaged in aconfocal arrangement. A linearly polarized laser excitation sourcepreferentially excites fluorescent target molecules with transitionmoments aligned parallel to the incident polarization vector. Theresultant fluorescence is collected and directed into two channels thatmeasure the intensity of the fluorescence polarized both parallel andperpendicular to that of the excitation beam. With these twomeasurements, the fluorescence anisotropy, r, can be determined from theequation: r=(Intensity parallel−Intensity perpendicular)/(Intensityparallel+2(Intensity perpendicular)) where the I terms indicateintensity measurements parallel and perpendicular to the incidentpolarization. Fluorescence anisotropy detection of fluorescent moleculeshas been described. Accordingly, fluorescence anisotropy can be coupledto numerous fluorescent labels as have been described herein and as havebeen described in the art.

In some embodiments, microbe can be detected through use of fluorescenceresonance energy transfer (FRET). Fluorescence resonance energy transferrefers to an energy transfer mechanism between two fluorescentmolecules. A fluorescent donor is excited at its fluorescence excitationwavelength. This excited state is then nonradiatively transferred to asecond molecule, the fluorescent acceptor. Fluorescence resonance energytransfer may be used within numerous configurations to detect capturedmicrobe. For example, in some embodiments, a first labeling molecule canbe labeled with a fluorescent donor and second labeling molecule can belabeled with a fluorescent acceptor. Accordingly, such labeled first andsecond labeling molecules can be used within competition assays todetect the presence and/or concentration of microbe in a sample.Numerous combinations of fluorescent donors and fluorescent acceptorscan be used for detection.

In some embodiments, a microbe can be detected through use ofpolynucleotide analysis. Examples of such methods include, but are notlimited to, those based on polynucleotide hybridization, polynucleotideligation, polynucleotide amplification, polynucleotide degradation, andthe like. Methods that utilize intercalation dyes, fluorescenceresonance energy transfer, capacitive deoxyribonucleic acid detection,and nucleic acid amplification have been described, for example, in U.S.Pat. Nos. 7,118, 910 and 6,960,437; herein incorporated by reference).Such methods can be adapted to provide for detection of one or moremicrobe nucleic acids. In some embodiments, fluorescence quenching,molecular beacons, electron transfer, electrical conductivity, and thelike can be used to analyze polynucleotide interaction. Such methods areknown and have been described, for example, in Jarvius, DNA Tools andMicrofluidic Systems for Molecular Analysis, Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 161,ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006, ISBN: 91-554-6616-8;Singh-Zocchi et al, Proc. Natl. Acad. Sci, 100:7605-7610 (2003); Wang etal. Anal. Chem, 75:3941-3945 (2003); and Fan et al, Proc. Natl. Acad.Sci, 100:9134-9137 (2003) and in U.S. Pat. Nos. 6,958,216; 5,093,268;and 6,090,545, the content of all of which is incorporated herein byreference. In some embodiments, the polynucleotide analysis is bypolymerase chain reaction (PCR). The fundamentals of PCR are well-knownto the skilled artisan, see, e.g. McPherson, et al., PCR, A PracticalApproach, IRL Press, Oxford, Eng. (1991), hereby incorporated byreference.

In some embodiments, a metabolic assay is used to determine the relativenumber of microbes in a sample compared to a control. As will beapparent to one of ordinary skill in the art any metabolic indicatorthat can be associated with cells can be used, such as but not limitedto, turbidity, fluorescent dyes, and redox indicators such as, but notlimited to, Alamar Blue, MTT, XTT, MTS, and WST. Metabolic indicatorscan be components inherent to the cells or components added to theenvironment of the cells. In some embodiments, changes in or the stateof the metabolic indicator can result in alteration of ability of themedia containing the sample to absorb or reflect particular wavelengthsof radiation.

Exemplary metabolic assays include, but are not limited to, ATPLuminescence, reactive oxygen species (ROS) assays, Resazurin assays,Luminol, MTT-metabolic assays, and the like. Further, as one of skill inthe art is well aware, kits and methods for carrying out metabolicassays are commercially available. For example,2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG),ATP Determination Kit, AMPLEX® Red Galactose/Galactose Oxidase AssayKit, AMPLEX® Red Glucose/Glucose Oxidase Assay Kit , AMPLEX® RedGlutamic Acid/Glutamate Oxidase Assay Kit, AMPLEX® Red HydrogenPeroxide/Peroxidase Assay Kit, AMPLEX® Red Monoamine Oxidase Assay Kit,AMPLEX® Red Neuraminidase (Sialidase) Assay Kit, AMPLEX® RedPhosphatidylcholine-Specific Phospholipase C Assay Kit, AMPLEX® RedSphingomyelinase Assay kit, AMPLEX® Red Uric Acid/Uricase Assay Kit,AMPLEX® Red Xanthine/Xanthine Oxidase Assay Kit, THIOLTRACKER™ Violet(Glutathione Detection Reagent), THIOLTRACKER™ Violet (GlutathioneDetection Reagent), and VYBRANT® Cell Metabolic Assay Kit fromInvitrogen; Adenosine 5′-triphospahte (ATP) Luminescence Assay Kit(ENLITEN® from Promega; ATPLITE™ from PerkinElmer Life Sciences; ATPBioluminescence Assay kit HS II from Boehringer Mannheim, Germany;Adenosine 5′-triphosphate (ATP) Luminescence Assay Kit from EMDMillipore; Reactive Oxygen Species (ROS) Assays from Cell BioLabs, Inc.;Cellular Reactive Oxygen Species Detection Assay Kit from ABCAM®; hROSDetection Kit from Cell Technology, Inc.; and ABTS Antioxidant AssayKit, ORAC Antioxidant Assay Kit, OxiSelect HORAC Activity Assay Kit,OxiSelect In vitro ROS/RNS Assay Kit (Green Fluorescence), OxiSelectIntracellular ROS Assay Kit (Green Fluorescence), OxiSelect ORACActivity Assay Kit, OxiSelect Total Antioxidant Capacity (TAC) AssayKit, and Total Antioxidant Capacity Assay Kit from BioCat.

In some embodiments, microbes isolated from or remained bound onmicrobe-targeting article can be labeled with nucleic acid barcodes forsubsequent detection and/or multiplexing detection. Nucleic acidbarcoding methods for detection of one or more analytes in a sample arewell known in the art.

In other embodiments, the captured microbe can be analyzed and/ordetected in the capture chamber or capture and visualization chamber ofa rapid microbe diagnostic device described in the Int. Pat. App. No.Int. Pat. App. No. WO 2011/091037, filed Jan. 19, 2011. Alternatively,the captured microbe can be recovered (i.e., removed) and analyzedand/or detected.

In some embodiments, the captured microbe is recovered and analyzedand/or detected using a particle on membrane assay as described in U.S.Pat. No. 7,781,226, content of which is incorporated herein byreference. A particle on membrane assay as described in U.S. Pat. No.7,781,226 can be operably linked with a rapid microbe diagnostic deviceof the Int. Pat. App. No. Int. Pat. App. No. WO 2011/091037 to reducethe number of sample handling steps, automate the process and/orintegrate the capture, separation and analysis/detection steps into amicrofluidic device.

In some embodiments, microbe capture, separation and analysis can bedone using a hybrid microfluidic SPR and molecular imagining device asdescribed in U.S. Pat. App. Pub. No. US 2011/0039280.

In some embodiments, the microbe capture, separation and analysis usingthe microbe-targeting molecules disclosed herein can be done by an assayor device described, for example, in PCT Application No.PCT/US2011/021603 filed Jan. 19, 2011, No. PCT/US2012/047201 filed Jul.18, 2012, and No. PCT/US2013/028409 filed Feb. 28, 2013, and U.S.Provisional Application No. 61/788570 filed Mar. 15, 2013, No.61/772,436 filed Mar. 4, 2013, No. 61/673,071 filed Jul. 18, 2013, andNo. 61/772,360 filed Mar. 4, 2013, contents of all of which areincorporated herein by reference.

In some embodiments, the processes or assays described herein can detectthe presence or absence of a microbe and/or identify a microbe in a testsample in less than 24 hours, less than 12 hours, less than 10 hours,less than 8 hours, less than 6 hours, less than 4 hours, less than 3hours, less than 2 hours, less than 1 hour, or lower. In someembodiments, the processes or assays described herein can detect thepresence or absence of a microbe and/or identify a microbe in a testsample in less than 6 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 1 hour, or lower.

Optional additional analyses or treatment—culturing: In some embodimentsof any aspects described herein, the assay or process can furthercomprise culturing any microbe bound on the microbe-targeting article(e.g., microbe-targeting microparticles) for a period of time. In suchembodiments, the microbe bound on the microbe-targeting article canexpand in population by at least about 10% after culturing for a periodof time.

In some embodiments, the microbe bound on the microbe-targeting article(e.g., microbe-targeting microparticle) can be cultured for a period oftime, e.g., at least about 15 mins, at least about 30 mins, at leastabout 1 hour, at least about 2 hours, at least about 3 hours, at leastabout 6 hours, at least about 9 hours, at least about 12 hours, at leastabout 18 hours, at least about 24 hours or longer. In some embodiments,the microbe bound on the microbe-targeting article can be cultured forat least about 30 mins to at least about 3 hours.

In some embodiments, the number of microbes bound on themicrobe-targeting article after culturing for a certain period of timecan be increased or expanded by at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, as compared to thenumber of the microbes originally bound on the microbe-targetingarticle. In some embodiments, the number of microbes bound on themicrobe-targeting article after culturing for a certain period of timecan be increased or expanded by at least about 1.5-fold, at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 50-fold, at least about100-fold, at least about 500-fold, at least about 1000-fold, at leastabout 10000-fold, at least about 100000-fold, as compared to the numberof the microbes originally bound on the microbe-targeting article.

In some embodiments, the microbes bound on the microbe-targetingarticles can be cultured on a microbe-compatible culture medium, e.g.,plated on an agar plate or cultured in LB broth. One of skill in the artwill readily recognize microbial culture techniques, including, but notlimited to, the use of incubators and/or equipment used to provide agentle agitation, e.g., rotator platforms, and shakers, if necessary,e.g., to prevent the cells from aggregation without subjecting them to asignificant shear stress and provide aerial agitation.

The microbes can remain bound on the microbe-article during detection oradditional analyses described herein or they can be detached, eluted offor removed from a microbe-targeting article prior to detection oradditional analyses described herein. In some embodiments where thebound microbes are desired to be detached, eluted off or removed from amicrobe-targeting article, the microbe-binding molecules of themicrobe-targeting article can be further contacted with a low pH buffer,e.g., a pH buffer less than 6, less than 5, less than 4, less than 3,less than 2, less than 1 or lower. In some embodiments, a low pH bufferthat does not cause precipitation of a chelating agent, if present, canbe used. In one embodiment, a low pH buffer can be arginine. In anotherembodiment, a low pH buffer can be pyrophosphate.

In some embodiments of any aspects described herein, the microbe-bindingmolecules of the microbe-targeting article can be further contacted witha low pH buffer and a chelating agent. In some embodiments, the contactof the microbe-binding molecules of the microbe-targeting article withthe low pH buffer and the chelating agent can be concurrent orsequentially. In one embodiment, the microbe-binding molecules of themicrobe-targeting substrate can be further contacted with arginine(e.g., 2 M) with EDTA or EGTA at pH 4.4.

The isolated microbes can then be used for analyses described earlier oradditional treatment, e.g., expansion in culture, antibiotic sensitivitytesting, sequencing and/or DNA or RNA analysis.

Optional additional analyses or treatment—antibiotic sensitivity orsusceptibility testing: In some embodiments of any aspects describedherein, the process or assay described herein can further comprisesubjecting the microbes bound on the microbe-targeting article or theexpanded cultures of microbes isolated from the microbe-targetingarticle to one or more antibiotics. The response of the microbe to anantibiotic can then be evaluated with any known methods in the art,e.g., by measuring the viability of microbes. Thus, an appropriateantibiotic can be identified for treatment of an infection caused by amicrobe, even though the specific species of the microbe bound onto themicrobe-targeting substrate is initially unknown. Additional details foruse of engineered microbe-targeting molecules described herein inantibiotic sensitivity testings can be found, e.g., in U.S. Prov. App.Nos. 61/604,878 filed Feb. 29, 2012 and 61/647,860 filed May 16, 2012,and PCT Application No. PCT/US2013/028409 filed Feb. 28, 2013, contentof all of which is incorporated herein by reference in their entireties.

Any processes or steps described herein can be performed by a module ordevice. While these are discussed as discrete processes, one or more ofthe processes or steps described herein can be combined into one systemfor carrying out the assays of any aspects described herein.

Exemplary Embodiments of Methods for Diagnosing or Locating a MicrobialInfection or Contamination

In general, embodiments of the assays or processes of any aspectsdescribed herein can be used to detect the presence or absence of amicrobe or microbial matter in a test sample or in situ (e.g., where themicrobe actually resides, e.g., in a water reservoir or on a workingsurface). For example, in some embodiments, a test sample, e.g.,obtained from a subject or an environmental source, or an environmentalsurface can be contacted with engineered microbe-binding molecules orengineered microbe-binding articles described herein, such that anymicrobes, if present, in the test sample or environmental surface can becaptured by the engineerd microbe-binding molecules or engineeredmicrobe-binding articles e.g., using any embodiments of the exemplaryprocess described above. In some embodiments, the captured microbesbound on the engineered microbe-binding molecules or microbe-bindingarticles can then be subjected to different analyses as described above,e.g., for identifying a microbe genus or species such as by immunoassay(e.g., using antibodies to a specific microbe), mass spectrometry, PCR,etc. In alternative embodiments where the engineered microbe-bindingmolecules comprise an imaging agent (e.g., a bubble, a liposome, asphere, a diagnostic contrast agent or a detectable label describedherein), the binding of the microbes to the engineered microbe-bindingmolecules can be detected in situ for identification of localizedmicrobial infection or contamination, and also allow localized treatmentof the infection or contamination.

In some embodiments, the assays or processes described herein can beused to diagnose or locate a microbial infection in situ in a subject.For example, engineered microbe-targeting microbeads comprising animaging agent (e.g., the engineered microbe-targeting microbeads can belinked to an imaging agent, e.g., a bubble, a liposome, a sphere, adiagnostic contrast agent or a detectable label described herein) can beadministered to a subject, either systemically (e.g., by injection), orlocally. In such embodiments, the engineered microbe-targetingmicrobeads comprising an imaging agent can be used to identify and/orlocalize pockets of localized microbial infection (e.g., in a tissue) inthe subject and optionally allow localized treatment of the microbialinfection, which is described in the section “Exemplary Compositions andMethods for Treating and/or Preventing a Microbial Infection” below.

For example, there is a strong need for more rapid and/or effectivediagnostic methods for distinguishing gram-positive microbe fromgram-negative microbes, which can permit physicians to initiate anappropriate drug therapy early on, rather than starting with asub-optimal or a completely ineffective antibiotic. A delay in treatmentof a microbial infection can significantly affect the treatment outcome,and can be sometimes fatal.

Thus, in some embodiments, the assays or processes described herein canbe used to distinguish a protein gram-positive microbe fromgram-negative microbe in a test sample.

Accordingly, exemplary methods of determining the presence or absence ofa gram-positive microbe infection in a subject are also provided herein.For example, the method can comprise contacting at least a first volumeor portion of a test sample with a microbe-targeting molecule or articledescribed herein and detecting binding of a microbe.

In some embodiments, the method can further comprise contacting at leasta second volume or portion of the test sample with at least one secondmicrobe-targeting molecule r a carrier scaffold conjugated to the same,wherein the second microbe-targeting molecule comprises at least onefirst domain wherein the CRP domain is replaced by a microbe-bindingdomain of microbe-binding domain protein which is not CRP. Thus, thesecond microbe-targeting molecule comprises at least one first domaincomprising at least a portion of a microbe-binding domain of amicrobe-binding protein, wherein the microbe-binding protein is not aCRP; a second domain, as described in this disclosure; and a linkerconjugating the first and the second domains. Microbe-binding domainsthat do not comprise CRP are described elsewhere in the disclosure.Exemplary second microbe-targeting molecules are described, for example,in PCT Application No. PCT/US2011/021603 filed Jan. 19, 2011 and No.PCT/US2012/047201, filed Jul. 18, 2012, and U.S. Provisional ApplicationNo. 61/691,983 filed Aug. 22, 2012, contents of all of which areincorporated herein by reference in their entireties. In someembodiments, the second microbe-targeting molecule comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 9,11, 12, and 23-27.

Generally, detection of binding with the CRP comprisingmicrobe-targeting molecules but not with the second secondmicrobe-targeting molecule can be indicative of presence of agram-positive microbe in the sample. Detection of binding with thesecond second microbe-targeting molecule but not with the CRP comprisingmicrobe-targeting molecules can be indicative of presence of agram-negative microbe in the sample. Detection of binding with the theCRP comprising microbe-targeting molecules and the second secondmicrobe-targeting molecule but not with the CRP comprisingmicrobe-targeting molecules can be indicative of presence of agram-negative microbe in the sample or both a gram-negative microbe anda gram-positive microbe in the sample.

In some embodiments, the method can further comprise administering orprescribing to the subject an antimicrobial agent when the subject isdetected with an infection. Some exemplary antimicrobial agents include,but are not limited to, penicillin, methicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, flucloxacillin, vancomycin, and anycombinations thereof.

Without wishing to be bound by theory, some embodiments of theengineered microbe-binding molecules can be used to opsonize a microbe,which is then cleared out by an innate immune response. In someembodiments, the microbe-targeting molecules can be a more potentopsonin of a microbe. Accordingly, in some embodiments, when the subjectis diagnosed with a microbial infection using the methods describedherein, the subject can be administered or prescribed with a compositioncomprising at least one engineered microbe-binding molecule describedherein. Without limitations, the methods of any aspects described hereincan be used to diagnose a microbe that is resistant to at least one, atleast two, at least three, at least four or more antibiotics.

In some embodiment, the assay disclosed herein can be performed using a“dipstick” format. By way of example only, a microbe-binding dipstick ortest strip can be brought into contact with a test sample (e.g., a bloodsample) from a patient or a subject, and incubated for a period of time,e.g., at least about 15 seconds, at least about 30 seconds, at leastabout 1 min, at least about 2 mins, at least about 5 mins, at leastabout 10 mins, at least about 15 mins, at least about 30 mins, at leastabout 1 hour or more. In some embodiments, the incubated dipstick ortest strip can then be incubated in a blocking agent (e.g., BSA, normalserum, casesin, non-fat dry milk, and/or any commercially-availableblocking agents to minimize non-specific binding). Depending ondifferent embodiments of the engineered microbe-targeting molecules, insome embodiments, the microbe-binding dipstick or test strip aftercontact with a test sample (e.g., a blood sample) can be furthercontacted with at least one additional agent to facilitate detection ofpathogen, and/or to increase specificity of the pathogen detection. Forexample, some embodiments of the dipstick or test strip after contactwith a test sample (e.g., a blood sample) can be further contacted witha detectable label that is conjugated to a molecule that binds to amicrobe and/or microbial matter. Examples of such molecules can include,but are not limited to, one or more embodiments of the engineeredmicrobe-targeting molecule described herein, an antibody specific forthe microbes or pathogens to be detected, a protein, a peptide, acarbohydrate or a nucleic acid that is recognized by the microbes orpathogens to be detected, and any combinations thereof.

In some embodiments, the readout of the microbe-binding dipsticks and/ortest strips can be performed in a system or device, e.g., a portabledevice. The system or device can display a signal indicating thepresence or the absence of a microbial infection in a test sample,and/or the extent of the microbial infection.

In one embodiment, the assay can be used for detecting or imaging anidus of infection in vivo. For example, a subject can be administered amicrobe-targeting molecule disclosed herein, wherein themicrobe-targeting molecule comprises a detectable label; and scanningthe subject using diagnostic imaging. Without limitations, thediagnostic imaging is selected from the group consisting of radiography,magnetic resonance imaging (MRI), Positron emission tomography (PET),Single-photon emission computed tomography (SPECT, or less commonly,SPET), Scintigraphy, ultrasound, CAT scan, photoacoustic imaging,thermography, linear tomography, poly tomography, zonography,orthopantomography (OPT or OPG), computed Tomography (CT) or ComputedAxial Tomography (CAT scan), and any combinations thereof.

Exemplary Compositions and Methods for Treating and/or Preventing aMicrobial Infection

The binding of microbes to engineered microbe-targeting molecules canfacilitate isolation and removal of microbes and/or microbial matterfrom an infected area. Accordingly, another aspect provided hereinrelate to compositions for treating and/or preventing a microbialinfection or microbial contamination comprising one or more engineeredmicrobe-targeting molecules or microbe-targeting substrates (e.g.,microbe-targeting magnetic microbeads) described herein.

In some embodiments, the composition can further comprise at least onesecond microbe-targeting molecule, wherein the second microbe-targetingmolecule comprises at least one first domain wherein the CRP domain isreplaced by a microbe-binding domain of microbe-binding domain proteinwhich is not CRP. Thus, the second microbe-targeting molecule comprisesat least one first domain comprising at least a portion of amicrobe-binding domain of a microbe-binding protein, wherein themicrobe-binding protein is not a CRP; a second domain, as described inthis disclosure; and a linker conjugating the first and the seconddomains. Microbe-binding domains that do not comprise CRP are describedelsewhere in the disclosure. Exemplary second microbe-targetingmolecules are described, for example, in PCT Application No.PCT/US2011/021603 filed Jan. 19, 2011 and No. PCT/US2012/047201, filedJul. 18, 2012, and U.S. Provisional Application No. 61/691,983 filedAug. 22, 2012, contents of all of which are incorporated herein byreference in their entireties. In some embodiments, the secondmicrobe-targeting molecule comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 9, 11, 12, and 23-27

In some embodiments, the composition can be formulated for treatingand/or preventing a microbial infection or a microbial contaminationpresent in an environmental surface. The term “environmental surface” asused herein refers to any surface and/or body of an environment or anobject. The environmental object can be a non-living object or a livingobject, e.g., a botanical plant. Examples of an environmental surfacecan include, but is not limited to, a medical device, an implantabledevice, a surface in a hospital or clinic (e.g., an operating room or anintensive-care unit), a machine or working surface for manufacturing orprocessing food or pharmaceutical products (e.g., drugs, therapeuticagents or imaging agents), a cell culture, a water treatment plant, awater reservoir and a botanical plant.

In some embodiments, the composition can be formulated for treatingand/or preventing microbial infection in a body fluid of a subject,e.g., blood. While in some embodiments, the engineered microbe-targetingmolecules of the composition described herein can capture microbesand/or microbial matter in a circulating body fluid, e.g., blood, inother embodiments, the engineered microbe-targeting molecules canopsonize a microbe and/or microbial matter such that the microbe and/ormicrobial matter can be recognized by an innate immune system forclearance.

Alternatively, the engineered microbe-targeting molecules can localize amicrobe and can thus prevent it from spreading, e.g., deeper into awound. In particular, the inventors have demonstrated that S. aureus canstrongly bind to some embodiments of the engineered microbe-targetingmolecules (e.g., microbe-binding magnetic microbeads) due to thepresence of both carbohydrate patterns and protein A on its microbialsurface capable of independent binding to the engineeredmicrobe-targeting molecules. Thus, in some embodiments, the engineeredmicrobe-targeting molecules can be used to localize a microbe load,which can then be easily removed from an infected area. In someembodiments, the microbead can be labeled for specific imaging ofinfected sites. For SPECT imaging the tracer radioisotopes typicallyused such as iodine-123, technetium-99m, xenon-133, thallium-201, andfluorine-18 can be used. Technetium 99m can be used for scintigraphicassay. Iodine-derived or other radioopaque contrast agents can also beincorporated in the beads for radiographic or CT-scan imaging. The useof paramagnetic or superparamagnetic microbeads can be used for magneticresonance imaging as contrast agents to alter the relaxation times ofatoms within a nidus of infection. In another embodiment, themicrospheres can be fluorescently dyed and applied to a surgical woundto determine the extension of an infectious process. This can be usefulfor assisting the surgeon in distinguishing between infected and healthytissues during debridment surgeries for osteomyelitis, cellulitis orfasciitis.

Accordingly, another aspect provided herein related to compositions fortreating and/or preventing a microbial infection in a tissue of asubject. In some embodiments, the composition comprises at least oneengineered microbe-targeting molecule as described herein. In someembodiments, the amount of the engineered microbe-targeting moleculesand/or microbe-targeting substrates present in the composition issufficient to reduce the growth and/or spread of the microbe in thetissue of the subject. The phrase “reducing the growth and/or spread ofthe microbe in the tissue” as used herein refers to reducing the numberof colonies of the microbe and/or movement of the microbe in the tissue.In some embodiments, the engineered microbe-targeting molecule cancapture and localize a microbe present in a tissue such that the numberof colonies of the microbe in the tissue can be reduced by at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, up to and including 100%, as compared toin the absence of the engineered microbe-targeting molecule. In someembodiments, the engineered microbe-targeting molecule can capture andlocalize a microbe present in a tissue such that the number of coloniesof the microbe in the tissue can be reduced by at least about 1.5-fold,at least about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 6-fold, at least about 7-fold, atleast about 8-fold, at least about 9-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold or more, as compared to inthe absence of the engineered microbe-targeting molecules. In oneembodiment, the binding of the engineered microbe-targeting moleculeswith a microbe (e.g., S. aureus) reduces the number of colonies by atleast about 4-fold to at least about 6-fold (e.g., at least about5-fold), as compared to in the absence of the engineeredmicrobe-targeting molecules, after a period of at least about 12 hours,at least about 16 hours or at least about 24 hours.

In other embodiments, the engineered microbe-targeting molecule cancapture and localize a microbe present in a tissue such that themovement of the microbe within the tissue (e.g., in terms of a distancetraveled deeper into the tissue and/or area of spread from the infectedsite) can be reduced by at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, up to andincluding 100%, as compared to in the absence of the engineeredmicrobe-targeting molecule. In some embodiments, the engineeredmicrobe-targeting molecule can capture and localize a microbe present ina tissue such that the movement of the microbe within the tissue (e.g.,in terms of a distance travelled deeper into the tissue and/or area ofspread from the infected site) can be reduced by at least about1.5-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold or more, ascompared to in the absence of the engineered microbe-targeting molecule.

In some embodiments, the composition can further comprise at least oneof an antimicrobial agent and a drug delivery vehicle. For example, insome embodiments, the composition can further comprise at least 1, atleast 2, at least 3, at least 4, at least 5 or more antimicrobialagents. In some embodiments, the composition can further comprise one ora plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 500, 1000 or more) delivery vehicles. In someembodiments, the composition can further comprise a combination of atleast one (including at least 2, at least 3, at least 4, at least 5 ormore) antimicrobial agent and at least one (including 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more)drug delivery vehicle. As used herein, the term “drug delivery vehicle”generally refers to any material that can be used to carry an activeagent to a target site. Examples of drug delivery vehicles includes, butare not limited to, a cell, a peptide particle, a polymeric particle, adendrimer, a vesicle, a liposome, a hydrogel, a nucleic acid scaffold,an aptamer, and any combinations thereof,

In some embodiments where a drug delivery vehicle is included, anengineered microbe-targeting molecule and/or an antimicrobial agent canbe dispersed within (e.g., encapsulated or embedded in) a drug deliveryvehicle and/or coated on a surface of the drug delivery vehicle.

In some embodiments where the composition includes at least oneantimicrobial agent, the antimicrobial agent can be present as aseparate entity from the engineered microbe-targeting molecule and/or itcan be fused with at least one engineered microbe-targeting molecule,e.g., by genetic modification and/or chemical conjugation.

The term “antimicrobial agent” as used herein refers to any entity withantimicrobial activity, i.e. the ability to inhibit or reduce the growthand/or kill a microbe, e.g., by at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 90% or more, ascompared to in the absence of an antimicrobial agent. An antimicrobialagent can be, for example, but not limited to, a silver nanoparticle, asmall molecule, a peptide, a peptidomimetics, an antibody or a fragmentthereof, a nucleic acid, an enzyme (e.g., an antimicrobialmetalloendopeptidase such as lysostaphin), an aptamer, a drug, anantibiotic, a chemical or any entity that can inhibit the growth and/orkill a microbe. Examples of an antimicrobial peptide that can beincluded in the composition described herein, include, but are notlimited to, mefloquine, venturicidin A, antimycin, myxothiazol,stigmatellin, diuron, iodoacetamide, potassium tellurite hydrate,aDL-vinylglycine, N-ethylmaleimide, L-allyglycine, diaryquinoline,betaine aldehyde chloride, acivcin, psicofuraine, buthioninesulfoximine, diaminopemelic acid, 4-phospho-D-erythronhydroxamic acid,motexafin gadolinium and/or xycitrin or modified versions or analoguesthereof.

In some embodiments, an antimicrobial agent included in the compositioncan be an antibiotic. As used herein, the term “antibiotic” is artrecognized and includes antimicrobial agents naturally produced bymicroorganisms such as bacteria (including Bacillus species),actinomycetes (including Streptomyces) or fungi that inhibit growth ofor destroy other microbes, or genetically-engineered thereof andisolated from such natural source. Substances of similar structure andmode of action can be synthesized chemically, or natural compounds canbe modified to produce semi-synthetic antibiotics. Exemplary classes ofantibiotics include, but are not limited to, (1) β-lactams, includingthe penicillins, cephalosporins monobactams, methicillin, andcarbapenems; (2) aminoglycosides, e.g., gentamicin, kanamycin, neomycin,tobramycin, netilmycin, paromomycin, and amikacin; (3) tetracyclines,e.g., doxycycline, minocycline, oxytetracycline, tetracycline, anddemeclocycline; (4) sulfonamides (e.g., mafenide, sulfacetamide,sulfadiazine and sulfasalazine) and trimethoprim; (5) quinolones, e.g.,ciprofloxacin, norfloxacin, and ofloxacin; (6) glycopeptides (e.g.,vancomycin, telavancin, teicoplanin); (7) macrolides, which include forexample, erythromycin, azithromycin, and clarithromycin; (8) carbapenems(e.g., ertapenem, doripenem, meropenem, and imipenem); (9)cephalosporins (e.g., cefadroxil, cefepime, and ceftobiprole); (10)lincosamides (e.g., clindamycin, and lincomycin); (11) monobactams(e.g., aztreonam); (12) nitrofurans (e.g., furazolidone, andnitrofurantoin); (13) Penicillins (e.g., amoxicillin, and Penicillin G);(14) polypeptides (e.g., bacitracin, colistin, and polymyxin B); and(15) other antibiotics, e.g., ansamycins, polymycins, carbacephem,chloramphenicol, lipopeptide, and drugs against mycobacteria (e.g., theones causing diseases in mammals, including tuberculosis (Mycobacteriumtuberculosis) and leprosy (Mycobacterium leprae), and any combinationsthereof.

Additional exemplary antimicrobial agent can include, but are notlimited to, antibacterial agents, antifungal agents, antiprotozoalagents, antiviral agents, and any mixtures thereof.

Exemplary antibacterial agents include, but are not limited to,Acrosoxacin, Amifioxacin, Amoxycillin, Ampicillin, Aspoxicillin,Azidocillin, Azithromycin, Aztreonam, Balofloxacin, lc Benzylpenicillin,Biapenem, Brodimoprim, Cefaclor, Cefadroxil, Cefatrizine, Cefcapene,Cefdinir, Cefetamet, Cefmetazole, Cefprozil, Cefroxadine, Ceftibuten,Cefuroxime, Cephalexin, Cephalonium, Cephaloridine, Cephamandole,Cephazolin,Cephradine, Chlorquinaldol, Chlortetracycline, Ciclacillin,Cinoxacin, Ciprofloxacin, Clarithromycin, Clavulanic Acid, Clindamycin,Clofazimine, Cloxacillin, Danofloxacin, Dapsone, Demeclocycline,Dicloxacillin, Difloxacin, Doxycycline, Enoxacin, Enrofloxacin,Erythromycin, Fleroxacin, Flomoxef, Flucloxacillin, Flumequine,Fosfomycin, Isoniazid, Levofloxacin, Mandelic Acid, Mecillinam,Metronidazole, Minocycline, Mupirocin, Nadifloxacin, Nalidixic Acid,Nifuirtoinol, Nitrofurantoin, Nitroxoline, Norfloxacin, Ofloxacin,Oxytetracycline, Panipenem, Pefloxacin, Phenoxymethylpenicillin,Pipemidic Acid, Piromidic Acid, Pivampicillin, Pivmecillinam,Prulifloxacin, Rufloxacin, Sparfloxacin, Sulbactam, Sulfabenzamide,Sulfacytine, Sulfametopyrazine, Sulphacetamide, Sulphadiazine,Sulphadimidine, Sulphamethizole, Sulphamethoxazole, Sulphanilamide,Sulphasomidine, Sulphathiazole, Temafioxacin, Tetracycline, Tetroxoprim,Tinidazole, Tosufloxacin, Trimethoprim, and phramceutically acceptablesalts or esters thereof.

Exemplary antifungal agents include, but are not limited to, Bifonazole,Butoconazole, Chlordantoin, Chlorphenesin, Ciclopirox Olamine,Clotrimazole, Eberconazole, Econazole, Fluconazole, Flutrimazole,Isoconazole, Itraconazole, Ketoconazole, Miconazole, Nifuroxime,Tioconazole, Terconazole, Undecenoic Acid, and pharmaceuticallyacceptable salts or esters thereof.

Exemplary antiprotozoal agents include, but are not limited to,Acetarsol, Azanidazole, Chloroquine, Metronidazole, Nifuratel,Nimorazole, Omidazole, Propenidazole, Secnidazole, Sineflngin,Tenonitrozole, Temidazole, Tinidazole, and pharmaceutically acceptablesalts or esters thereof.

Exemplary antiviral agents include, but are not limited to, Acyclovir,Brivudine, Cidofovir, Curcumin, Desciclovir, 1-Docosanol, Edoxudine, gQFameyclovir, Fiacitabine, Ibacitabine, Imiquimod, Lamivudine,Penciclovir, Valacyclovir, Valganciclovir, and pharmaceuticallyacceptable salts or esters thereof.

In some embodiments, the antimicrobial agent can include silver presentin any form, e.g., a nanoparticle, a colloid, a suspension, powder, andany combinations thereof.

In some embodiments, the composition can be used to treat and/or preventan infection caused by any microbe described herein. In one embodiment,the composition can be used to treat and/or prevent an infection causedby S. aureus.

In some embodiments, the composition can be used to treat and/or preventan infection caused by a microbe that is resistant to at least one, atleast two, at least three, at least four or more antimicrobial agentsdescribed herein. In one embodiment, the composition can be used totreat and/or prevent an infection caused by a microbe that is resistantto at least one, at least two, at least three, at least four or moreantibiotics described herein. For example, in one embodiment, thecomposition can be used to treat and/or prevent an infection caused bymethicillin-resistant S. aureus. In another embodiment, the compositioncan be used to treat and/or prevent an infection caused byvancomycin-resistant S. aureus.

Exemplary antimicrobial applications and/or products: The compositionsdescribed herein can be formulated or configured for differentapplications and/or products such antimicrobial products. In someembodiments, the composition described herein can be formulated aspharmaceutical compositions as described below, e.g., for therapeutictreatment as an antibiotic or antiseptic.

Wound dressings: In some embodiments, the composition described hereincan be formulated for topical application, e.g., in wounds, lesions orabscesses. By way of example only, in some embodiments, a plurality ofengineered microbe-targeting molecules can be blended with, attached toor coated on a wound dressing, for example, but not limited to, abandage, an adhesive, a gauze, a film, a gel, foam, hydrocolloid,alginate, hydrogel, paste (e.g., polysaccharide paste), a spray, agranule and a bead.

In some embodiments, the wound dressing can include an additionalantimicrobial agent described herein and/or an antiseptic chemical,e.g., boracic lint and/or medicinal castor oil.

In one embodiment, a plurality of engineered microbe-targeting molecules(e.g., microbe-targeting microparticles or microbe-targeting magneticmicrobeads) can be attached or coated onto a wound dressing such as abandage or an adhesive. When such wound dressing is applied to a woundor a lesion, any microbe (e.g., S. aureus) and/or microbial matterpresent in the wound or lesion can bind and localized to the wounddressing. Thus, regular replacement of the wound dressing can remove themicrobe from the wound or lesion and thus prevent the microbe frommoving deeper into the wound or lesion for further infection.

In one embodiment, a plurality of engineered microbe-targeting molecules(e.g., microbe-targeting microparticles or microbe-targeting magneticmicrobeads) can be formulated into a wound dressing spray, which can behandy and used anywhere, e.g., during a transportation on an emergencyvehicle. When the wound dressing spray containing the microbe-targetingmagnetic microbeads, the microbe-targeting magnetic microbeads withbound microbes (e.g., S. aureus) can be removed from the wound with amagnetic field gradient before re-application of the spray.

Debridement fluids or sprays: In some embodiments, the compositiondescribed herein can be formulated as part of a debridement fluid(optionally with suspended particulates that are abrasive to a lesionarea). In some embodiments, the composition described herein can beformulated as part of a debridement spray. As used herein, the term“debridement” generally refers to complete or partial removal of asubject's dead, damaged, and/or infected tissue to improve the healingpotential of the remaining healthy and/or non-infected tissue. By way ofexample only, a plurality of engineered microbe-targeting molecules(e.g., microbe-targeting microparticles or magnetic microbeads) can besuspended in a debridement fluid or spray, e.g., for use in anorthopedic procedure. The debridement fluid or spray containing theengineered microbe-targeting molecules can be applied to a lesion, anabscess or a wound, where the engineered microbe-targetingmicroparticles or magnetic microbeads can capture a microbe (e.g., S.aureus) and/or microbial matter from the lesion, abscess or wound. Thedebridement fluid or spray can then be removed from the applied site byvacuum, or suction. In some embodiments, the debridement fluid or spraycontaining the engineered microbe-targeting magnetic microbeads can bealso removed from the applied site by exposing the applied site to amagnetic field gradient, which can pull or attract the appliedmicrobe-targeting magnetic microbeads out from the applied site.

Medical device coating: In some embodiments, the composition describedherein can be coated on a surface of a medical device, e.g., a fluiddelivery device such as hollow fibers, tubing or a spiral mixer in anextracorporeal device, or an implantable device such as an indwellingcatheter, chip or scaffold. By way of example only, a plurality ofengineered microbe-targeting molecules can be coated or conjugated to asurface of a fluid delivery device such that when a fluid (e.g., blood)flows through the fluid delivery device coated with engineeredmicrobe-targeting molecules, any microbe (e.g., S. aureus) and/ormicrobial matter present in the fluid (e.g., blood) can be extractedtherefrom, thus reducing the chance of a microbial infection. In anotherembodiment, a plurality of engineered microbe-targeting molecules coatedon a medical device can comprise a detectable label, e.g., a “smartlabel” described herein, which can provide a detectable signal when anymicrobe (e.g., S. aureus) binds to a surface of the medical device,indicating that the medical device has been contaminated and/orinfected, and thus is not appropriate for use or implantation.

The disclosure further provides methods for removing a microbe and/ormicrobial matter from a target area comprising contacting the targetarea with at least one composition described herein. As removal of amicrobe and/or microbial matter from an infected area can treat and/orprevent a microbial infection or microbial contamination, providedherein also include methods for treating and/or preventing a microbialinfection or microbial contamination in a target area. An exemplarymethod comprises contacting the target area with a compositioncomprising the engineered microbe-targeting molecule disclosed herein.The target area can be anywhere, e.g., an environmental surface or in abody of a subject (e.g., body fluid, and/or tissue). In someembodiments, the method comprises contacting the tissue of the subjectwith any embodiments of the composition described herein. In someembodiments, the tissue can have an open wound, a lesion or an abscess.

In one embodiment, the composition can be formulated for use as a wounddressing described herein.

As the engineered microbe-targeting molecules can localize a microbe(e.g., S. aureus) for easier removal of the microbe from the tissue, insome embodiments, the method can further comprise replacing thepreviously-applied composition in contact with the tissue with a freshcomposition after a period of time. For example, depending on thecondition of the microbial infection and/or specific compositions, thepreviously-applied composition can be replaced every 1 hour, every 2hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every8 hours, every 10 hours, every 12 hours, every 16 hours, every 24 hoursor longer.

In some embodiments, the method can further comprise administering anadditional treatment to the tissue. Exemplary additional treatments caninclude, but are not limited to, a negative-pressure treatment, avacuum-assisted debridement, administration of an antimicrobial agent,or any combinations thereof.

Without limitations, the compositions and/or methods of any aspectsdescribed herein can be used to treat and/or prevent a microbialinfection or contamination in vitro, in situ or in vivo. In someembodiments, the compositions and/or methods of any aspects describedherein can be used to treat and/or prevent a microbial infection orcontamination in a fluid or on any surface, including, but not limitedto, a tissue surface, a solid substrate surface, e.g., a medical devicesurface, an environmental surface, or food.

Additionally, in some embodiments where the composition comprises atleast one engineered microbe-targeting molecule conjugated to adetectable label described herein or an imaging agent, can be used toimage an infection in situ, e.g., in a subject or on an environmentalsurface.

The disclosure also provides a method for delivering or concentrating ananti-microbial agent at a nidus of infection. Generally, the nidus iscontacted with a composition of comprising at least onemicrobe-targeting molecule disclosed herein and an anti-microbial agent.The microbial agent can be covalently or non-covalently linked with themicrobe-targeting molecule. In some embodiments, the anti-microbialagent can be encompassed in a particle covalently or non-covalentlylined with the microbe-targeting molecule.

In some embodiments, the composition further comprises at least onesecond microbe-targeting molecule, wherein the second microbe-targetingmolecule comprises at least one first domain wherein the CRP domain isreplaced by a microbe-binding domain of microbe-binding domain proteinwhich is not CRP. Thus, the second microbe-targeting molecule comprisesat least one first domain comprising at least a portion of amicrobe-binding domain of a microbe-binding protein, wherein themicrobe-binding protein is not a CRP; a second domain, as described inthis disclosure; and a linker conjugating the first and the seconddomains. Microbe-binding domains that do not comprise CRP are describedelsewhere in the disclosure. Exemplary second microbe-targetingmolecules are described, for example, in PCT Application No.PCT/US2011/021603 filed Jan. 19, 2011 and No. PCT/US2012/047201, filedJul. 18, 2012, and U.S. Provisional Application No. 61/691,983 filedAug. 22, 2012, contents of all of which are incorporated herein byreference in their entireties. In some embodiments, the secondmicrobe-targeting molecule comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 9, 11, 12, and 23-27.

Pharmaceutical Compositions

Some embodiments of the engineered microbe-targeting molecules can beused for therapeutic purposes. For administration to a subject in needthereof, engineered microbe-targeting molecules described herein can beprovided in pharmaceutically acceptable compositions. Accordingly, inyet another aspect, provided herein is a pharmaceutical compositioncomprising at least one engineered microbe-targeting molecule describedherein, and a pharmaceutically acceptable carrier.

When the engineered microbe-targeting molecules are used as therapeuticsin vivo, the second domain or the linker can be further modified tomodulate the effector function such as antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). By wayof example only, the Fc region for use as a second domain can mediateADCC and CDC. In ADCC, the Fc region can generally bind to Fc receptorson the surface of immune effector cells such as natural killers andmacrophages, leading to the phagocytosis or lysis of a targeted cell. InCDC, the Fc region can generally trigger the complement cascade at thecell surface to kill the targeted cell. Accordingly, modulating effectorfunctions can be achieved by engineering the Fc region to eitherincrease or decrease their binding to the Fc receptors on the surface ofthe immune effector cells or the complement factors. For example,numerous mutations within a Fc region for modulating ADCC and CDC arewell known to a skilled artisan, e.g., see Armour K L. et al. (1999) EurJ Immmunol 29: 2613-2624; Shields R L. et al. (2001) J Biol Chem. 276:6591-6604; Idusogie E E. et al. (2001) J Immunol. 166: 2571-2575;Idusogie E E. et al. (2000) J Immunol. 155: 1165-1174; and Steurer W. etal. (1995) J Immunol. 155: 1165-1674. In one embodiment, the amino acidasparagine (N) at the residue 82 of the SEQ ID NO. 6 can be mutated toaspartic acid (D), e.g., to remove the glycosylation of Fc and thus, inturn, reduce ADCC and CDC functions.

In some embodiments, the pharmaceutical composition further comprises atleast one second microbe-targeting molecule, wherein the secondmicrobe-targeting molecule comprises at least one first domain whereinthe CRP domain is replaced by a microbe-binding domain ofmicrobe-binding domain protein which is not CRP. Thus, the secondmicrobe-targeting molecule comprises at least one first domaincomprising at least a portion of a microbe-binding domain of amicrobe-binding protein, wherein the microbe-binding protein is not aCRP; a second domain, as described in this disclosure; and a linkerconjugating the first and the second domains. Microbe-binding domainsthat do not comprise CRP are described elsewhere in the disclosure.Exemplary second microbe-targeting molecules are described, for example,in PCT Application No. PCT/US2011/021603 filed Jan. 19, 2011 and No.PCT/US2012/047201, filed Jul. 18, 2012, and U.S. Provisional ApplicationNo. 61/691,983 filed Aug. 22, 2012, contents of all of which areincorporated herein by reference in their entireties. In someembodiments, the second microbe-targeting molecule comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 9,11, 12, and 23-27.

Depending on the selected administration route, the compositions orpreparations can be in any form, e.g., a tablet, a lozenge, asuspension, a free-flowing powder, an aerosol, and a capsule. The term“pharmaceutically acceptable,” as used herein, refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” refers toa pharmaceutically-acceptable material, composition or vehicle foradministration of an active agent described herein. Pharmaceuticallyacceptable carriers include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like which are compatible with the activity ofthe active agent and are physiologically acceptable to the subject. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (i) sugars, such as lactose, glucose and sucrose; (ii)starches, such as corn starch and potato starch; (iii) cellulose, andits derivatives, such as sodium carboxymethyl cellulose,methylcellulose, ethyl cellulose, microcrystalline cellulose andcellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin;(vii) lubricating agents, such as magnesium stearate, sodium laurylsulfate and talc; (viii) excipients, such as cocoa butter andsuppository waxes; (ix) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (x)glycols, such as propylene glycol; (xi) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such asethyl oleate and ethyl laurate; (xiii) agar; (xiv) buffering agents,such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid;(xvi) pyrogen-free water; (xvii) isotonic saline; (xviii) Ringer'ssolution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi)polyesters, polycarbonates and/or polyanhydrides; (xxii) bulking agents,such as polypeptides and amino acids (xxiii) serum component, such asserum albumin, HDL and LDL; (xxiv) C2-C12 alcohols, such as ethanol; and(xxv) other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.For compositions or preparations described herein to be administeredorally, pharmaceutically acceptable carriers include, but are notlimited to pharmaceutically acceptable excipients such as inertdiluents, disintegrating agents, binding agents, lubricating agents,sweetening agents, flavoring agents, coloring agents and preservatives.Suitable inert diluents include sodium and calcium carbonate, sodium andcalcium phosphate, and lactose, while corn starch and alginic acid aresuitable disintegrating agents. Binding agents may include starch andgelatin, while the lubricating agent, if present, will generally bemagnesium stearate, stearic acid or talc. If desired, the tablets may becoated with a material such as glyceryl monostearate or glyceryldistearate, to delay absorption in the gastrointestinal tract.

Pharmaceutically acceptable carriers can vary in a preparation describedherein, depending on the administration route and formulation. Thecompositions and preparations described herein can be delivered via anyadministration mode known to a skilled practitioner. For example, thecompositions and preparations described herein can be delivered in asystemic manner, via administration routes such as, but not limited to,oral, and parenteral including intravenous, intramuscular,intraperitoneal, intradermal, and subcutaneous. In some embodiments, thecompositions and preparations described herein are in a form that issuitable for injection. In other embodiments, the compositions andpreparations described herein are formulated for oral administration.

When administering parenterally, a composition and preparation describedherein can be generally formulated in a unit dosage injectable form(solution, suspension, emulsion). The compositions and preparationssuitable for injection include sterile aqueous solutions or dispersions.The carrier can be a solvent or dispersing medium containing, forexample, water, cell culture medium, buffers (e.g., phosphate bufferedsaline), polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof. In someembodiments, the pharmaceutical carrier can be a buffered solution (e.g.PBS).

An oral composition can be prepared in any orally acceptable dosage formincluding, but not limited to, tablets, capsules, emulsions and aqueoussuspensions, dispersions and solutions. Commonly used carriers fortablets include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added to tablets. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.Liquid preparations for oral administration can also be prepared in theform of a dry powder to be reconstituted with a suitable solvent priorto use.

The compositions can also contain auxiliary substances such as wettingor emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON′S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation. With respect tocompositions described herein, however, any vehicle, diluent, oradditive used should have to be biocompatible with the active agentsdescribed herein. Those skilled in the art will recognize that thecomponents of the compositions should be selected to be biocompatiblewith respect to the active agent. This will present no problem to thoseskilled in chemical and pharmaceutical principles, or problems can bereadily avoided by reference to standard texts or by simple experiments(not involving undue experimentation).

In some embodiments, the compositions and preparations described hereincan be formulated in an emulsion or a gel. Such gel compositions andpreparations can be implanted locally to a diseased tissue region of asubject.

For in vivo administration, the compositions or preparations describedherein can be administered with a delivery device, e.g., a syringe.Accordingly, an additional aspect described herein provides for deliverydevices comprising at least one chamber with an outlet, wherein the atleast one chamber comprises a pre-determined amount of any compositiondescribed herein and the outlet provides an exit for the compositionenclosed inside the chamber. In some embodiments, a delivery devicedescribed herein can further comprise an actuator to control release ofthe composition through the outlet. Such delivery device can be anydevice to facilitate the administration of any composition describedherein to a subject, e.g., a syringe, a dry powder injector, a nasalspray, a nebulizer, or an implant such as a microchip, e.g., forsustained-release or controlled release of any composition describedherein.

In some embodiments of the products described herein, themicrobe-targeting microparticles described herein itself can be modifiedto control its degradation and thus the release of active agents. Insome embodiments, the engineered microbe-targeting molecules,microbe-targeting microparticles and/or microbe-targeting cellsdescribed herein can be combined with other types of delivery systemsavailable and known to those of ordinary skill in the art. They include,for example, polymer-based systems such as polylactic and/orpolyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates,polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/orcombinations thereof. Microcapsules of the foregoing polymers containingdrugs are described in, for example, U.S. Pat. No. 5,075,109. Otherexamples include nonpolymer systems that are lipid-based includingsterols such as cholesterol, cholesterol esters, and fatty acids orneukal fats such as mono-, di- and triglycerides; hydrogel releasesystems; liposome-based systems; phospholipid based- systems; silasticsystems; peptide based systems; or partially fused implants. Specificexamples include, but are not limited to, erosional systems in which thecomposition is contained in a form within a matrix (for example, asdescribed in U.S. Pat. Nos. 4,452, 775, 4,675,189, 5,736,152, 4,667,014,4,748,034 and -29 5,239,660), or diffusional systems in which an activecomponent controls the release rate (for example, as described in U.S.Pat. Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). Theformulation may be as, for example, microspheres, hydrogels, polymericreservoirs, cholesterol matrices, or polymeric systems. In someembodiments, the system may allow sustained or controlled release of thecomposition to occur, for example, through control of the diffusion orerosion/degradation rate of the formulation containing the composition.In addition, a pump-based hardware delivery system can be used todeliver one or more embodiments of the compositions or preparationsdescribed herein. Use of a long-term sustained release formulations orimplants can be particularly suitable for treatment of some infections.Long-term release, as used herein, means that a formulation or animplant is made and arranged to deliver compositions or preparationsdescribed herein at a therapeutic level for at least 30 days, or atleast 60 days. In some embodiments, the long-term release refers to aformulation or an implant being configured to deliver an active agent ata therapeutic level over several months.

Kits

Kits for capturing, detecting and/or determining the presence or absenceof a microbe and/or microbial matter in a sample are also providedherein. In some embodiments, the kit can comprise: (a) one or morecontainers containing a population of engineered microbe-targetingmolecules described herein; and (b) at least one reagent. In theseembodiments, a user can generate their own microbe-targeting substratesby conjugating the provided engineered microbe-targeting molecules totheir desired substrate, e.g., using any art-recognized conjugationchemistry and/or methods described herein. In such embodiments, thereagent can include, but is not limited to, a coupling agent forconjugation of engineered microbe-targeting molecules to a substrate. Insome embodiments, the kit can further comprise one or more substrates(e.g., microbeads such as magnetic microbeads) to which the engineeredmicrobe-targeting molecules described herein are conjugated. In suchembodiments, a user can further modify the surface chemistry of theprovided substrate prior to conjugation of the engineeredmicrobe-targeting molecules to the substrate.

In other embodiments, the kit can provide microbe-targeting substratesthat are ready for use. Accordingly, in these embodiments, the kit cancomprise: (a) one or more microbe-targeting substrates described herein;and (b) at least one reagent. In some embodiments, the microbe-targetingsubstrate can include one or more microbe-binding dipsticks, e.g., asdescribed herein. In other embodiments, the microbe-targeting substratecan include a population of microbe-targeting microbeads (including, butnot limited to, polymeric microbeads and magnetic microbeads). In someembodiments, the microbe-targeting substrate can include a population ofmicrobe-targeting magnetic microbeads. The microbe-targeting microbeadsor microbe-targeting magnetic microbeads can be provided in one or moreseparate containers, if desired. In some embodiments, the population ofthe microbe-targeting microbeads or magnetic microbeads contained in oneor more containers can be lyophilized.

In some embodiments, a kit disclosed herein can further comprise atleast one second microbe-targeting molecule or a secondmicrobe-targeting molecule conjugated to a carrier scaffold, wherein thesecond microbe-targeting molecule comprises at least one first domainwherein the CRP domain is replaced by a microbe-binding domain ofmicrobe-binding domain protein which is not CRP. Thus, the secondmicrobe-targeting molecule comprises at least one first domaincomprising at least a portion of a microbe-binding domain of amicrobe-binding protein, wherein the microbe-binding protein is not aCRP; a second domain, as described in this disclosure; and a linkerconjugating the first and the second domains. Microbe-binding domainsthat do not comprise CRP are described elsewhere in the disclosure.Exemplary second microbe-targeting molecules are described, for example,in PCT Application No. PCT/US2011/021603 filed Jan. 19, 2011, and No.PCT/US2012/047201, filed Jul. 18, 2012, contents of both of which areincorporated herein by reference in their entireties. In someembodiments, the second microbe-targeting molecule comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 9,11, 12, and 23-27

In some embodiments of any aspects of the kits described herein, thepopulation of the microbeads or microbe-targeting microbeads cancomprise at least one distinct subset of the microbeads ormicrobe-targeting microbeads, respectively. For example, each distinctsubset of the microbeads or microbe-targeting microbeads can be providedin a separate container. In some embodiments, the distinct subset of themicrobeads or microbe-targeting microbeads can have a size. In someembodiments, the distinct subset of microbe-targeting microbeads cancomprise on their surfaces a different density of engineeredmicrobe-targeting molecules from the rest of the population. In theseembodiments, two or more subsets of the microbe-targeting microbeshaving different sizes and/or different coating density of theengineered microbe-binding molecules can be used to detect anddifferentiate microbes of different classes and/or sizes, e.g.,employing the methods described herein. In some embodiments, thedistinct subset of microbe-targeting substrates, e.g., microbe-targetingmicrobeads, can comprise a different carbohydrate recognition domainfrom the others.

In some embodiments of any aspects of the kits described herein, thesubstrates (e.g., microbeads) or microbe-targeting substrates (e.g.,microbe-targeting microbeads) can further comprise a detection label. Byway of example only, depending on the choice of detection methods, eachdistinct subset of the microbeads can comprise a unique detection labelor the same detection label. For example, if each distinct subset of themicrobe-targeting microbeads is used in a different sampling well, thesame detection label can be used on the microbe-targeting microbeads.However, if it is desirable to detect multiple differentmicrobe-targeting microbeads in the same well, it is preferably to haveeach distinct subset of microbe-targeting microbeads comprising adistinct detection label.

Detectable labels suitable for use in any kits provided herein includeany composition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Anyart-recognized detectable labels or the ones described herein can beincluded in the kits described herein.

Means of detecting such labels are well known to those of skill in theart and exemplary detection methods are described herein. For example,radiolabels can be detected using photographic film or scintillationcounters, fluorescent markers can be detected using a photo-detector todetect emitted light. Enzymatic labels are typically detected byproviding the enzyme with an enzyme substrate and detecting the reactionproduct produced by the action of the enzyme on the enzyme substrate,and calorimetric labels can be detected by visualizing the coloredlabel.

In some embodiments of any aspects described herein, the kits canfurther comprise one or more containers containing a population ofdetectable labels, wherein the detectable label is conjugated to amolecule. In some embodiments, at least one of the containers cancontain a distinct population of detectable labels.

In some embodiments, at least one of the containers can contain adistinct population of the molecule-detectable label conjugate asdescribed earlier. The distinct population of the molecule-detectablelabel conjugate can contain a unique molecule with the detectable labelsame as others, or a conjugate comprising a distinct detectable label(e.g., a unique fluorescent molecule) and a distinct molecule. As eachdistinct detectable label can identify the associated protein,conjugates comprising a distinct detectable label associated with adistinct molecule can allow detecting in a single sample at least two ormore distinct populations of the engineered microbe-targeting substrates(e.g., microbe-targeting magnetic microbeads); for example, eachdistinct population of the engineered microbe-targeting magneticmicrobeads can bind to a distinct genus or species or type/size of amicrobe. In alternative embodiments, the molecule-detectable labelconjugates in each of the containers can comprise the same detectablelabel. For example, the detectable label can comprise an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) that produces a colorchange in the presence of an enzyme substrate. In such embodiments, thekit can further comprise one or more containers containing an enzymesubstrate that changes color in the presence of the enzyme.

In one embodiment, the microbe-targeting article provided in the kit caninclude a dipstick or test strip or membrane containing one or moreengineered microbe-targeting molecules, e.g., microbe-binding dipstickor membrane described herein. In this embodiment, the kit can comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200 or moremicrobe-binding dipsticks or test strips described herein. These kitscomprising the microbe-binding dipsticks or test strips can be used as adiagnostic or probe for a microbe anywhere, e.g., at home, in clinics orhospitals, on emergency vehicles, in outdoor environments, in foodprocessing plants, and anywhere in need of microbe capture and/ordetection.

In some embodiments, each microbe-targeting article described herein,e.g., each microbe-binding dipstick or membrane, can be individuallypackaged to maintain their sterility. In some embodiments, two or moreproducts (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or moreproducts such as microbe-binding dipsticks or membranes) can be packagedinto one single unit. In such embodiments, users can sterilize anyunused products after opening, e.g., with UV radiation, hightemperature, gamma-radiation, ethylene oxide sterilization or any otherknown methods that would not significantly affect the activity of theengineered microbe-targeting molecules for microbe detection.

In other embodiments, the microbe-targeting article provided in the kitcan include a population of microbe-targeting microparticles. In someembodiments, the microbe-targeting microparticles can be lyophilized.

Depending on the configuration/combination of the molecule-detectablelabel conjugates provided in the kit, different populations of themicrobe-targeting microparticles can be mixed together with a testsample in a single reaction, or different populations each can beapplied separately to different aliquots of the same test sample. Aftercontacting the test sample with the microbe-targeting microbeads ormagnetic microbeads, any microbes or pathogens recognized by themicrobe-targeting molecules will bind to the microbe-targetingmicroparticles.

In some embodiments, the kit can further comprise at least one bloodcollection container or any equivalent sample container or chamber,including at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20 blood collection containers or equivalent sample containersor chambers. In some embodiments, the population of themicrobe-targeting microbeads or magnetic microbeads can be pre-loaded inat least one blood collection container. In some embodiments, the bloodcollection container can further comprise an anti-coagulant agentdescribed herein. In some embodiments, a blood sample can be directlyadded to such blood collection container containing a population of themicrobe-targeting articles for carrying out a microbe detection assay,e.g., as described herein. An ordinary artisan will readily appreciatethat some embodiments of the microbe-targeting articles (withoutmagnetic properties) described herein can also be applicable for theassay. For example, instead of using a magnet to collect themicrobe-targeting magnetic microparticless after contact with a testsample (e.g., a blood sample), the microbe-targeting article (withoutmagnetic properties) can also be collected, e.g., by filtration,centrifugation or any other methods known in the art.

In some embodiments where the kits comprise microbe-targeting magneticmicrobeads, the kits can further comprise a magnet adapted for use withthe assay for isolation of the microbe-targeting magnetic microbeadsfrom a test sample. For example, if the assay is carried out in a bloodcollection tube, the magnet can be adapted for use with the bloodcollection tube, e.g., a magnet can be designed to be a magnet collarsurrounding the blood collection tube to immobilize or isolate themicrobe-targeting magnetic microbeads from a test sample or an assaybuffer.

In any aspects of the kits provided herein, the kits can furthercomprise a portable readout machine or device, e.g., to determine anddisplay the signal produced from the assay performed with the kit. Forexample, the readout machine or device can detect a colorimetric signaland/or a fluorescent signal produced from the assay of pathogendetection performed with the kits described herein.

In any aspects of the kits described herein, the kits can furtherinclude a reference for comparison with a readout determined from a testsample. An exemplary reference can be a strip or a chart showingdifferent colors corresponding to various extents or degrees of amicrobial infection.

Depending on different embodiments of the engineered microbe-targetingmolecules and/or products provided in the kits, some embodiments of anyaspects of the kits described herein can further comprise an additionalagent. For example, in some embodiments where the engineeredmicrobe-targeting molecules present on the substrate are unlabeled, thekit can further comprise one or more containers containing a populationof detectable labels described earlier, each of which is conjugated to atargeting agent specific for a microbe, e.g., without limitations, oneor more embodiments of an engineered microbe-targeting molecule or afragment thereof, an antibody specific for at least one microbe (e.g.,antibodies specific for Gram-positive microbes such as anti-LTAantibodies, antibodies specific for Gram-negative microbes such asanti-LPS antibodies, or antibodies specific for fungus , and anycombinations thereof). The use of an additional targeting agent specificfor a microbe conjugated to a detectable label can not only facilitatethe detection of microbes or pathogens, but can also increase thespecificity of the detection for a microbe or a pathogen.

In any aspects of the kits provided herein, when the detection labelincludes an enzyme (e.g., horseradish peroxidase, alkaline phosphataseand any others commonly used for colorimetric detection), the kits canfurther comprise one or more containers containing an enzyme substratethat produces a color change in the presence of the enzyme. One of skillin the art can readily recognize an appropriate enzyme substrate for anyart-recognized enzymes used for colorimetric detection. By way ofexample only, an exemplary substrate for alkaline phosphatase caninclude BCIP/NBT or PNPP (p-Nitrophenyl Phosphate, Disodium Salt); anexemplary substrate for horseradish peroxidase can include TMB.

In any aspects of the kits provided herein, the at least one reagent canbe a wash buffer, a dilution buffer, a stop buffer, e.g., to stop thecolor development, a buffer solution containing a chelating agentdescribed herein, or any combinations thereof. In one embodiment, atleast one of the reagents provided in the kit can include at least onebuffered solution containing a chelating agent. The chelating agent canbe used to chelate any ions (e.g., divalent ions) present in the testsamples or assay buffer, e.g., for inhibiting calcium-dependent bindingof certain microbes, but not others, to some embodiments of themicrobe-binding molecules described herein. Accordingly, such kit can beused to distinguish one microbe (e.g., S. aureus) from another (e.g., E.coli) in a test sample, e.g. employing some embodiments of the methoddescribed herein.

In any aspects of the kits provided herein, the kits can furthercomprise at least one microtiter plate, e.g., for performing thereaction and the detection.

In addition to the above mentioned components, any embodiments of thekits described herein can include informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the aggregates for the methods described herein. For example, theinformational material can describe methods for using the kits providedherein to perform an assay for pathogen or microbe capture and/ordetection. The kit can also include an empty container and/or a deliverydevice, e.g., which can be used to deliver a test sample to a testcontainer.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments, the kit can contain separate containers, dividersor compartments for each component and informational material. Forexample, each different component can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, acollection of the magnetic microbeads is contained in a bottle, vial orsyringe that has attached thereto the informational material in the formof a label.

In general, the kits described herein can be used to separate, remove,and/or detect a microbe present in a test sample. In some embodiments,the kits can be used to differentiate between different microbe species,classes, and/or sizes, by employing the methods and/or assays describedherein. By way of example only, some embodiments of the kits can be usedto detect the presence or absence of a gram-positive microbe in a testsample. Accordingly, some embodiments of the kits described herein canbe used to detect or determine the presence or absence of at least onegram-positive microbe in a test sample.

In some embodiments, the kits described herein can be used to screen apharmaceutical product (e.g., a drug, a therapeutic agent, or an imagingagent), or a medical device (including, but not limited to, implantabledevices) for the presence or absence of microbial matter (including, butnot limited to, endotoxins secreted by a microbe).

Test Sample

In accordance with various embodiments described herein, a test sampleor sample, including any fluid or specimen (processed or unprocessed),that is suspected of comprising a microbe and/or microbial matter can besubjected to an assay or method, kit and system described herein. Thetest sample or fluid can be liquid, supercritical fluid, solutions,suspensions, gases, gels, slurries, and combinations thereof. The testsample or fluid can be aqueous or non-aqueous.

In some embodiments, the test sample can be an aqueous fluid. As usedherein, the term “aqueous fluid” refers to any flowable water-containingmaterial that is suspected of comprising a microbe and/or microbialmatter.

In some embodiments, the test sample can include a biological fluidobtained from a subject. Exemplary biological fluids obtained from asubject can include, but are not limited to, blood (including wholeblood, plasma, cord blood and serum), lactation products (e.g., milk),amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid,bronchial aspirate, perspiration, mucus, liquefied feces, synovialfluid, lymphatic fluid, tears, tracheal aspirate, and fractions thereof.In some embodiments, a biological fluid can include a homogenate of atissue specimen (e.g., biopsy) from a subject.

In some embodiments, the biological fluid sample obtained from asubject, e.g., a mammalian subject such as a human subject or a domesticpet such as a cat or dog, can contain cells from the subject. In otherembodiments, the biological fluid sample can contain non-cellularbiological material, such as non-cellular fractions of blood, saliva, orurine, which can be used to measure plasma/serum biomarker expressionlevels.

The biological fluid sample can be freshly collected from a subject or apreviously collected sample. In some embodiments, the biological fluidsample used in the assays and/or methods described herein can becollected from a subject no more than 24 hours, no more than 12 hours,no more than 6 hours, no more than 3 hours, no more than 2 hours, nomore than 1 hour, no more than 30 mins or shorter.

In some embodiments, the biological fluid sample or any fluid sampledescribed herein can be treated with a chemical and/or biologicalreagent described herein prior to use with the assays and/or methodsdescribed herein. In some embodiments, at least one of the chemicaland/or biological reagents can be present in the sample container beforea fluid sample is added to the sample container. For example, blood canbe collected into a blood collection tube such as VACUTAINER®, which hasalready contained heparin. Examples of the chemical and/or biologicalreagents can include, without limitations, surfactants and detergents,salts, cell lysing reagents, anticoagulants, degradative enzymes (e.g.,proteases, lipases, nucleases, collagenases, cellulases, amylases), andsolvents such as buffer solutions.

In some embodiments, the test sample can include a fluid or specimenobtained from an environmental source, e.g., but not limited to, watersupplies (including wastewater), ponds, rivers, reservoirs, swimmingpools, soils, food processing and/or packaging plants, agriculturalplaces, hydrocultures (including hydroponic food farms), pharmaceuticalmanufacturing plants, animal colony facilities, and any combinationsthereof.

In some embodiments, the test sample can include a fluid (e.g., culturemedium) from a biological culture. Examples of a fluid (e.g., culturemedium) obtained from a biological culture includes the one obtainedfrom culturing or fermentation, for example, of single- or multi-cellorganisms, including prokaryotes (e.g., bacteria) and eukaryotes (e.g.,animal cells, plant cells, yeasts, fungi), and including fractionsthereof. In some embodiments, the test sample can include a fluid from ablood culture. In some embodiments, the culture medium can be obtainedfrom any source, e.g., without limitations, research laboratories,pharmaceutical manufacturing plants, hydrocultures (e.g., hydroponicfood farms), diagnostic testing facilities, clinical settings, and anycombinations thereof.

In some embodiments, the test sample can include a media or reagentsolution used in a laboratory or clinical setting, such as forbiomedical and molecular biology applications. As used herein, the term“media” refers to a medium for maintaining a tissue, an organism, or acell population, or refers to a medium for culturing a tissue, anorganism, or a cell population, which contains nutrients that maintainviability of the tissue, organism, or cell population, and supportproliferation and growth.

As used herein, the term “reagent” refers to any solution used in alaboratory or clinical setting for biomedical and molecular biologyapplications. Reagents include, but are not limited to, salinesolutions, PBS solutions, buffered solutions, such as phosphate buffers,EDTA, Tris solutions, and any combinations thereof. Reagent solutionscan be used to create other reagent solutions. For example, Trissolutions and EDTA solutions are combined in specific ratios to create“TE” reagents for use in molecular biology applications.

In some embodiments, the test sample can be a non-biological fluid. Asused herein, the term “non-biological fluid” refers to any fluid that isnot a biological fluid as the term is defined herein. Exemplarynon-biological fluids include, but are not limited to, water, saltwater, brine, buffered solutions, saline solutions, sugar solutions,carbohydrate solutions, lipid solutions, nucleic acid solutions,hydrocarbons (e.g. liquid hydrocarbons), acids, gasoline, petroleum,liquefied samples (e.g., liquefied samples), and mixtures thereof.

Exemplary Microbes or Pathogens

As used herein, the term “microbes” or “microbe” generally refers tomicroorganism(s), including bacteria, fungi, protozoan, archaea,protists, e.g., algae, and a combination thereof. The term “microbes”encompasses both live and dead microbes. The term “microbes” alsoincludes pathogenic microbes or pathogens, e.g., bacteria causingdiseases such as plague, tuberculosis and anthrax; protozoa causingdiseases such as malaria, sleeping sickness and toxoplasmosis; fungicausing diseases such as ringworm, candidiasis or histoplasmosis; andbacteria causing diseases such as sepsis. In some embodiments, themicrobe is a gram-positive microbe.

Microbe-induced diseases: In some other embodiments, the engineeredmicrobe-targeting molecules or articles, assays, products and kitsdescribed herein can be used to detect or bind to gram-positive microbeor associated microbial matter. Some exemplary gram-positive microbesinclude, but are not limited to, the genera Aerococcus, Bacillus,Bifdobacterium, Carcina, Clostridium, Corprococcus, Corynebacterium,Deinobacter, Deinococcus, Enterococcus, Erysipelothrix, Eubacterium,Gemella, Lactobacillus, Lactococcus, Leuconostoc, Listeria,Marinococcus, Micrococcus, Pediococcus, Peptococcus, Peptostreptococcus,Planococcus, Propionibacterium, Ruminococcus, Saccharococcus,Salinococcus, Staphylococcus, Staphylococcus, Stomatococcus,Streptococcus, Streptomyces, Trichococcus, and Vagococcus. Some specificgram-positive microbe species include, but are not limited to,Actinomyces spp., Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothennophilus, Bacillus subtilis, Bacillus thuringiensis,Bifidobacterium spp., Clostridium clostridiiforme, Clostridiumdifficile, Clostridium innocuum, Clostridium perfringens, Clostridiumramosum, Corynebacterium jeikeium, E. lentum, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Eubacterium aerofaciens,L. casei, L. plantarum, Lactobacillus acidophilus, Lactococcus lactis,Lactococcus spp., Leuconostoc spp., Listeria monocytogenes, Moraxellaspp. (including M. catarrhalis), Mycobacterium leprae , Mycobacteriumtuberculosis, P. asaccarolyticus, P. magnus, P. micros, P. prevotii, P.productus, Pediococcus, Peptostreptococcus anaerobius, Propionibacteriumacnes, Staphylococcus aureus, Staphylococcus aureus (MRSA),Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus lugdunensis, Staphylococcus saprophytics,Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis,Streptococcus lactis, Streptococcus mitis, Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus sangius, Streptococcus viridans,and Streptomyces lividans.

In some embodiments, the engineered microbe-targeting molecules orarticles, products, and kits described herein can be described hereincan be used to differentiate a gram-positive microbe from agram-negative microbe by employing the methods or assays describedherein.

One skilled in the art can understand that the engineeredmicrobe-targeting molecules or substrates, products and kits describedherein can be used to target any microorganism with a microbesurface-binding domain described herein modified for each microorganismof interest. A skilled artisan can determine the cell-surface proteinsor carbohydrates for each microorganism of interest using anymicrobiology techniques known in the art.

Biofilm: Accordingly, in some embodiments, the microbe-targetingmolecules or substrates, products and kits herein can be used to detectmicrobes and/or associated microbial matter present in a biofilm or totreat equipment surfaces to prevent or inhibit formation of a biofilm.For example, Listeria monocytogenes can form biofilms on a variety ofmaterials used in food processing equipment and other food and non-foodcontact surfaces (Blackman, J Food Prot 1996; 59:827-31; Frank, J FoodProt 1990; 53:550-4; Krysinski, J Food Prot 1992; 55:246-51; Ronner, JFood Prot 1993; 56:750-8). Biofilms can be broadly defined as microbialcells attached to a surface, and which are embedded in a matrix ofextracellular polymeric substances produced by the microorganisms.Biofilms are known to occur in many environments and frequently lead toa wide diversity of undesirable effects. For example, biofilms causefouling of industrial equipment such as heat exchangers, pipelines, andship hulls, resulting in reduced heat transfer, energy loss, increasedfluid frictional resistance, and accelerated corrosion. Biofilmaccumulation on teeth and gums, urinary and intestinal tracts, andimplanted medical devices such as catheters and prostheses frequentlylead to infections (Characklis W G. Biofilm processes. In: Characklis WG and Marshall K C eds. New York: John Wiley & Sons, 1990:195-231;Costerton et al., Annu Rev Microbiol 1995; 49:711-45). In someembodiments, the engineered microbe-targeting microparticles, e.g.,encapsulating a drug or a chemical for treatment of a biofilm, can besprayed on contaminated equipment surfaces. The bacteria present in thebiofilm bind to the microbe-targeting microparticles, which release thedrug to treat the bacteria for targeted drug delivery.

In addition, L. monocytogenes attached to surfaces such as stainlesssteel and rubber, materials commonly used in food processingenvironments, can survive for prolonged periods (Helke and Wong, J FoodProt 1994; 57:963-8). This would partially explain their ability topersist in the processing plant. Common sources of L. monocytogenes inprocessing facilities include equipment, conveyors, product contactsurfaces, hand tools, cleaning utensils, floors, drains, walls, andcondensate (Tomkin et al., Dairy, Food Environ Sanit 1999; 19:551-62;Welbourn and Williams, Dairy, Food Environ Sanit 1999; 19:399-401). Insome embodiments, the engineered microbe-targeting molecules can beconfigured to include a “smart label”, which is undetectable whenconjugated to the engineered microbe-targeting molecules, but produces acolor change when released from the engineered molecules in the presenceof a microbe enzyme. Thus, when a microbe binds to the engineeredmicrobe-targeting molecules, the microbe releases enzymes that releasethe detectable label from the engineered molecules. An observation of acolor change indicates a risk for bacteria contamination on a particularsurface, and thus some embodiments of the engineered microbe-targetingmolecules and products can be used for early detection of biofilmformation.

Plant microbes: In still further embodiments, the engineeredmicrobe-targeting molecules or substrates and products described hereincan be used to target plant microbes and/or associated microbial matter.Plant fungi have caused major epidemics with huge societal impacts.Examples of plant fungi include, but are not limited to, Phytophthorainfestans, Crimpellis perniciosa, frosty pod (Moniliophthora roreri),oomycete Phytophthora capsici, Mycosphaerella fijiensis, FusariumGanoderma spp fungi and Phytophthora. An exemplary plant bacteriumincludes Burkholderia cepacia. Exemplary plant viruses include, but arenot limited to, soybean mosaic virus, bean pod mottle virus, tobaccoring spot virus, barley yellow dwarf virus, wheat spindle streak virus,soil born mosaic virus, wheat streak virus in maize, maize dwarf mosaicvirus, maize chlorotic dwarf virus, cucumber mosaic virus, tobaccomosaic virus, alfalfa mosaic virus, potato virus X, potato virus Y,potato leaf roll virus and tomato golden mosaic virus.

Military and bioterrorism applications: In yet other embodiments, theengineered microbe-targeting molecules and product comprising thereofcan be used to detect or combat bioterror agents (e.g., B. Anthracis,and smallpox).

In accordance with some embodiments described herein, an engineeredmicrobe-binding molecule or microbe-binding substrate can be modified tobind to any of the microbes, e.g., the ones described herein, includingthe associated microbial matter (e.g., but not limited to, fragments ofcell wall, microbial nucleic acid and endotoxin).

Exemplary embodiments of the various aspects disclosed herein can bedescribed by one or more of the numbered paragraphs:

-   1. A microbe-targeting molecule comprising:    -   a. at least one first domain comprising at least a portion of a        c-reactive protein (CRP);    -   b. at least one second domain comprising at least a portion of a        domain selected from the group consisting of:        -   i. Fc region of an immunoglobulin;        -   ii. microbe-binding domain of a microbe-binding protein,            wherein the microbe-binding protein is not CRP;        -   iii. neck region of a lectin;        -   iv. a detectable label;        -   v. domain for conjugation to surface of a carrier scaffold;        -   vi. pattern recognition receptor domain of CRP; and        -   vii. any combinations of (i)-(vi); and    -   c. a linker conjugating the first and second domains.-   2. The microbe-targeting molecule of paragraph 1, wherein the first    domain is conjugated to N-terminus of the second domain.-   3. The microbe-targeting molecule of paragraph 1, wherein the second    domain is conjugated to N-terminus of the first domain.-   4. The microbe-targeting molecule of any of paragraphs 1-3, wherein    the molecule is a multimeric molecule.-   5. The microbe-targeting molecule of paragraph 4, wherein the    molecule is a pentamer.-   6. The microbe-targeting molecule of paragraph 4 or 5, wherein the    multimeric molecule is formed by interactions between the linkers    and/or the second domains of different molecules forming the    multimeric molecule.-   7. The microbe-targeting molecule of any of paragraphs 1-6, wherein    the first domain comprises the amino acid sequence selected from SEQ    ID NOs: 1-4 and 39.-   8. The microbe-targeting molecule of any of paragraphs 1-7, wherein    the detectable molecule is selected from the group consisting of    biotin, fluorophore, luminescent or bioluminescent marker, a    radiolabel, an enzyme, an enzyme substrate, a quantum dot, an    imaging agent, a gold particle, and any combinations thereof.-   9. The microbe-targeting molecule of paragraph 8, wherein the enzyme    is horseradish peroxide or alkaline phosphatase.-   10. The microbe-targeting molecule of any of paragraphs 1-9, wherein    the immunoglobulin is selected from the group consisting of IgA,    IgD, IgE, IgG, and IgM.-   11. The microbe-targeting molecule of any of paragraphs 1-11,    wherein the Fc region comprises at least one region selected from    the group consisting of a hinge region, a CH2 region, a CH3 region,    and any combinations thereof.-   12. The microbe-targeting molecule of any of paragraphs 1-12,    wherein the Fc region comprises at least one mutation.-   13. The microbe-targeting molecule of paragraph 13, wherein the    mutation is selected to: (i) increase biological half-life of the    engineered molecule; (ii) modulate anti-body dependent cell-mediated    cytotoxicity; and/or (iii) modulate complement dependent    cytotoxicity.-   14. The microbe-targeting molecule of any of paragraphs 1-13,    wherein the Fc region comprises the amino acid sequence SEQ ID NO:    5, 6, 7 or 42.-   15. The microbe-targeting molecule of any of paragraphs 1-14,    wherein the microbe-binding protein is a carbohydrate binding    protein and the microbe-binding domain is a carbohydrate recognition    domain (CRD) of the carbohydrate binding protein.-   16. The microbe-targeting molecule of paragraph 15, wherein the CRD    excludes at least one of complement and coagulation region of the    carbohydrate binding protein.-   17. The microbe-targeting molecule of paragraph 15 or 16, wherein    the CRD is from a lectin or ficolin.-   18. The microbe-targeting molecule of any of paragraphs 15-17,    wherein the CRD is from a Collectin.-   19. The microbe-targeting molecule of any of paragraphs 15-18,    wherein the CRD is from a mannose-binding lection (MBL).-   20. The microbe-targeting molecule of any of paragraphs 15-19,    wherein the CRD comprises the amino acid sequences SEQ ID NO: 8, 9,    11, or 12.-   21. The microbe-targeting molecule of any of paragraphs 1-19,    wherein the neck region comprises an amino acid sequence from neck    region of a lectin.-   22. The microbe-targeting molecule of any of paragraphs 1-20,    wherein the neck region comprises the amino acid sequence SEQ ID NO:    28, 29, 30, or 31.-   23. The microbe-targeting molecule of any of paragraphs 1-21,    wherein the second domain comprises at least a portion of Fc region    of an immunoglobulin and at least a portion of the neck region of a    lectin.-   24. The microbe-targeting molecule of paragraph 22, wherein the neck    region is between the Fc region and the first domain.-   25. The microbe-targeting molecule of any of paragraphs 1-23,    wherein the domain for conjugation to surface of a carrier scaffold    comprises an amino group, a N-substituted amino group, a carboxyl    group, a carbonyl group, an acid anhydride group, an aldehyde group,    a hydroxyl group, an epoxy group, a thiol, a disulfide group, an    alkenyl group, a hydrazine group, a hydrazide group, a semicarbazide    group, a thiosemicarbazide group, one partner of a binding pair, an    amide group, an aryl group, an ester group, an ether group, a    glycidyl group, a halo group, a hydride group, an isocyanate group,    an urea group, an urethane group, and any combinations thereof.-   26. The microbe-targeting molecule of any of paragraphs 1-24,    wherein the domain for conjugation to surface of the carrier    scaffold comprises the N-terminal amino acid sequence AKT (SEQ ID    NO: 32).-   27. The microbe-targeting molecule of any of paragraphs 1-25,    wherein the domain for conjugation to surface of the carrier    scaffold is present at a terminus end of the engineered molecule.-   28. The microbe-targeting molecule of any of paragraphs 1-26,    wherein the second domain comprises an amino acid sequence selected    from the group consisting of SEQ ID NOs: 5-12, 23-27, 32, 42, and    any combinations thereof.-   29. The microbe-targeting molecule of any of paragraphs 1-27,wherein    the microbe-targeting molecule comprises an amino acid sequence    selected from the group consisting of SEQ ID NOs: 33-38, 40, 41, 43,    44, and any combinations thereof.-   30. A pharmaceutical composition comprising a microbe-targeting    molecule of any of paragraphs 1-29 and a pharmaceutically acceptable    excipient or carrier.-   31. An article comprising at least one microbe-targeting molecule of    any of paragraphs 1-29 conjugated to a surface of a carrier    scaffold.-   32. The article of paragraph 31, wherein the carrier scaffold is    selected from the group consisting of a nucleic acid scaffold, a    protein scaffold, a lipid scaffold, a polymeric scaffold, a    dendrimer, a particle or bead, a nanotube, a microtiter plate, a    medical apparatus or implant, a microchip, a filtration device, a    membrane, a diagnostic strip, a dipstick, an extracorporeal device,    a spiral mixer, a hollow-fiber tube, a living cell, a biological    tissue or organ, magnetic material, hollow fiber, and any    combinations thereof.-   33. The article of paragraph 31 or 32, wherein the carrier scaffold    is a nano- or micro-particle.-   34. The article of any of paragraphs 31-33, wherein the carrier    scaffold is a magnetic particle, a florescent particle, a quantum    dot, a drug delivery vehicle, a dipstick, a paper strip, a membrane,    a hollow fiber tube, or a gold particle.-   35. The article of any of paragraphs 31-34, wherein the carrier    scaffold further comprises at least one area adapted for use as a    reference area.-   36. The article of any of paragraphs 31-35, wherein the carrier    scaffold is modified or functionalized.-   37. The article of any of paragraphs 31-36, wherein the carrier    scaffold is treated to reduce or inhibit adhesion of the carrier    scaffold to a biological molecule.-   38. The article of paragraph 37, wherein the biological molecule is    selected from the group consisting of blood cells and components,    proteins, nucleic acids, peptides, small molecules, therapeutic    agents, cells or fragments thereof, and any combinations thereof.-   39. The article of any of paragraphs 31-36, wherein the article    further comprises a detection label.-   40. The article of paragraph 39, wherein the detection label is    separate from the microbe-targeting molecule.-   41. The article of any of paragraphs 31-40, wherein the article    further comprises at least one second microbe-targeting molecule,    wherein the second microbe-targeting molecule comprises:    -   a. at least one first domain comprising at least a portion of a        microbe-binding domain of a microbe-binding protein, wherein the        microbe-binding protein is not CRP;    -   b. at least one second domain comprising at least a portion of a        domain selected from the group consisting of:        -   i. Fc region of an immunoglobulin;        -   ii. neck region of a lectin;        -   iii. a detectable label;        -   iv. domain for conjugation to surface of a carrier scaffold;            and        -   v. any combinations of (i)-(iv); and    -   c. a linker conjugating the first and second domains.-   42. An assay for determining the presence or absence of a microbe in    a test sample, the assay comprising:    -   (i) contacting a test sample with a microbe-targeting molecule        of any of paragraphs 1-29 or an article of any of paragraphs        31-40; and    -   (ii) detecting binding of a microbe to the microbe-targeting        molecule,    -   wherein a microbe is present in the test sample if binding is        detected.-   43. An assay for differentiating a gram positive microbe from a gram    negative microbe in a test sample, the assay comprising:    -   (i) contacting a test sample with a microbe-targeting molecule        of any of paragraphs 1-29 or an article of any of paragraphs        31-40; and    -   (ii) detecting binding of a microbe to the microbe-targeting        molecule,    -   wherein the gram positive microbe is selectively bound to the        microbe-targeting molecule, thereby differentiating the gram        positive microbe from the gram negative microbe in the test        sample.-   44. The assay of paragraph 42 or 43, wherein said detecting step    comprises an enzyme-linked immunosorbent assay (ELISA), a    fluorescent linked immunosorbent assay (FLISA), immunofluorescent    microscopy, hybridization, fluorescence in situ hybridization    (FISH), antibody-based imaging, a radiological detection assay, a    chemical detection assay, an enzymatic detection assay, an optical    detection assay, an electrochemical assay, cell culture, or any    combinations thereof.-   45. The assay of any of paragraphs 42-44, wherein said detecting    step comprises contacting the sample from step (i) with a    microbe-targeting molecule of any of paragraphs 1-29, wherein the    microbe-targeting molecule is conjugated with a detectable label.-   46. The assay of any of paragraphs 42-45, wherein the microbe-bound    microbe-targeting molecule is detectable without prior cell culture.-   47. The assay of any of paragraphs 42-46, wherein the contacting    step comprises flowing the test sample through a channel, wherein    the channel is coated with the microbe-targeting molecules.-   48. The assay of any of paragraphs 42-47, wherein the contacting    step comprises flowing the test sample and microbe-targeting    molecule through a channel.-   49. An assay for differentiating a gram positive microbe from a gram    negative microbe in a test sample, the assay comprising:    -   (i) contacting a first portion of a test sample with a        microbe-targeting molecule of any of paragraphs 1-29 or an        article of any of paragraphs 31-40 and detecting binding of a        microbe to the microbe-targeting molecule or the article; and    -   (ii) contacting a second portion of the test sample with a        second microbe-targeting molecule or an article comprising at        least one second microbe-targeting molecule conjugated to a        surface of a carrier scaffold, wherein the second        microbe-targeting comprises:        -   a. at least one first domain comprising at least a portion            of a microbe-binding domain of a microbe-binding protein,            wherein the microbe-binding protein is not CRP;        -   b. at least one second domain comprising at least a portion            of a domain selected from the group consisting of:            -   i. Fc region of an immunoglobulin;            -   ii. neck region of a lectin;            -   iii. a detectable label;            -   iv. domain for conjugation to surface of a carrier                scaffold; and            -   v. any combinations of (i)-(iv); and        -   c. a linker conjugating the first and second domains            wherein:    -   (a) binding in step (i) and not in step (ii) is indicative of a        gram-positive microbe in the sample;    -   (b) binding in step (ii) and not in step (i) is indicative of a        gram-negative microbe in the sample; and    -   (c) binding in both step (i) and step (ii) is indicative of a        gram-positive microbe in the sample or both a gram-positive        microbe and a gram-negative microbe in the sample.-   50. The assay of paragraph 49, wherein said detecting in step (i)    or (ii) comprises an enzyme-linked immunosorbent assay (ELISA), a    fluorescent linked immunosorbent assay (FLISA), immunofluorescent    microscopy, hybridization, fluorescence in situ hybridization    (FISH), antibody-based imaging, a radiological detection assay, a    chemical detection assay, an enzymatic detection assay, an optical    detection assay, an electrochemical assay, cell culture, or any    combinations thereof.-   51. The assay of paragraph 49 or 50, wherein said detecting in    step (i) comprises contacting the sample with a microbe-targeting    molecule of any of paragraphs 1-29, wherein the microbe-targeting    molecule is conjugated with a detectable label.-   52. The assay of any of paragraphs 49-51, wherein said detecting in    step (ii) comprises contacting the sample with the second    microbe-targeting molecule, wherein the microbe-targeting molecule    is conjugated with a detectable label.-   53. A kit comprising:    -   (i) one or more microbe-targeting molecules of any of paragraphs        1-29 or one or more articles of any of paragraphs 31-40; and    -   (ii) a reagent.-   54. A kit comprising:    -   (i) one or more microbe-targeting molecules of any of paragraphs        1-29 or one or more articles of any of paragraphs 31-40; and    -   (ii) one or more second microbe-targeting molecules or an        article comprising at least one second microbe-targeting        molecule conjugated to a surface of a carrier scaffold, wherein        the second microbe-targeting molecules comprise:        -   a. at least one first domain comprising at least a portion            of a microbe-binding domain of a microbe-binding protein,            wherein the microbe-binding protein is not CRP;        -   b. at least one second domain comprising at least a portion            of a domain selected from the group consisting of:            -   i. Fc region of an immunoglobulin;            -   ii. neck region of a lectin;            -   iii. a detectable label;            -   iv. domain for conjugation to surface of a carrier                scaffold; and            -   v. any combinations of (i)-(iv); and        -   c. a linker conjugating the first and second domains.-   55. A composition for treating and/or preventing a microbial    infection or a microbial contamination, the composition comprising    at least one microbe-targeting molecule of any of paragraphs 1-29 or    an article of any of paragraphs 31-40.-   56. The composition of paragraph 55, further comprising a second    microbe-targeting molecule or an article comprising at least one    second microbe-targeting molecule conjugated to a surface of a    carrier scaffold, wherein the second microbe-targeting molecule    comprises:    -   a. at least one first domain comprising at least a portion of a        microbe-binding domain of a microbe-binding protein, wherein the        microbe-binding protein is not CRP;    -   b. at least one second domain comprising at least a portion of a        domain selected from the group consisting of:        -   i. Fc region of an immunoglobulin;        -   ii. neck region of a lectin;        -   iii. a detectable label;        -   iv. domain for conjugation to surface of a carrier scaffold;            and        -   v. any combinations of (i)-(iv); and    -   c. a linker conjugating the first and second domains.-   57. The composition of paragraph 55 or 56, wherein the composition    is formulated for treating and/or preventing microbial infection or    contamination of a surface.-   58. The composition of paragraph 57, wherein the surface is selected    from the group consisting of a medical device, an implantable    device, hospital or clinic, a machine or working surface for    processing food or pharmaceutical products, a cell culture, a water    treatment plant, a water reservoir, a botanical plant, and any    combinations thereof.-   59. The composition of any of paragraphs 55-57, wherein the    composition is formulated for treating and/or preventing a microbial    infection in a subject.-   60. The composition of any of paragraphs 55-58, wherein the    composition is formulated for treating and/or preventing a microbial    infection in a tissue or a body fluid of a subject.-   61. The composition of any of paragraphs 55-59, further comprising    an antimicrobial agent.-   62. The composition of paragraph 60, wherein the antimicrobial agent    is conjugated with the microbe-targeting molecule.-   63. The composition of paragraph 60 or 61, further comprising a drug    delivery vehicle.-   64. The composition of paragraph 62, wherein at least one of the    microbe-targeting molecule or the antimicrobial agent is coated on a    surface of the drug delivery vehicle.-   65. The composition of paragraph 62 or 63, wherein the drug delivery    vehicle is selected from the group consisting of a peptide particle,    a polymeric particle, a dendrimer, a vesicle, a liposome, a    hydrogel, a nucleic acid scaffold, an aptamer, and any combinations    thereof.-   66. A method of inhibiting, preventing, and/or treating a microbial    infection or contamination in a target area, the method comprising    contacting the target area with a first composition of any of    paragraphs 55-65.-   67. A method of removing a microbe or microbial matter thereof from    a target area, the method comprising contacting the target area with    a first composition of any of paragraphs 55-65.-   68. The method of paragraph 66 or 67, wherein the target area    comprises an environmental surface.-   69. The method of paragraph 68, wherein the environmental surface is    selected from the group consisting of a medical device, an    implantable device, hospital or clinic, a machine or working surface    for processing food or pharmaceutical products, a cell culture, a    water treatment plant, a water reservoir, a botanical plant, and any    combinations thereof.-   70. The method of any of paragraphs 66-69, wherein the target area    is a tissue or a body fluid of a subject.-   71. The method of paragraph 70, further comprising administering an    additional treatment to the tissue.-   72. The method of paragraph 71, wherein the additional treatment    includes a negative-pressure treatment, a vacuum-assisted    debridement, administration of an antimicrobial agent, or any    combinations thereof.-   73. The method of any of paragraphs 66-72, further comprising    replacing the first composition in contact with the tissue with a    second composition of any of paragraphs 55-65 after a period of    time.-   74. A method comprising:    -   administering a therapeutic agent having activity against a        gram-positive microbe to a subject in need thereof, wherein the        therapeutic agent is selected based on the presence a        gram-positive microbe in a test sample from the subject, and        wherein the presence or absence of the gram-positive microbe in        the sample is determined by the assay of any of paragraphs        42-52.-   75. The method of paragraph 74, further comprising performing the    assay.-   76. A method for detecting or imaging a nidus of infection in vivo,    the method comprising:    -   administering to a subject a microbe-targeting molecule of any        of paragraphs 1-X, wherein the microbe-targeting molecule        comprises a detectable label; and    -   scanning the subject using diagnostic imaging.-   77. The method of paragraph 76, wherein diagnostic imaging is    selected from the group consisting of radiography, magnetic    resonance imaging (MRI), Positron emission tomography (PET),    Single-photon emission computed tomography (SPECT, or less commonly,    SPET), Scintigraphy, ultrasound, CAT scan, photoacoustic imaging,    thermography, linear tomography, poly tomography, zonography,    orthopantomography (OPT or OPG), computed Tomography (CT) or    Computed Axial Tomography (CAT scan), and any combinations thereof.-   78. A method of delivering or concentrating an anti-microbial agent    at a nidus of infection, the method comprising contacting the nidus    of infection with a composition of comprising at least one    microbe-targeting molecule of any of paragraphs 1-29 or an article    of any of paragraphs 31-40, wherein the composition further    comprises an anti-microbial agent.-   79. The method of paragraph 78, wherein said nidus of infection is    in vivo.-   80. The method of paragraph 78 or 79, wherein the composition    further comprises a second microbe-targeting molecule or an article    comprising at least one second microbe-targeting molecule conjugated    to a surface of a carrier scaffold, wherein the second    microbe-targeting molecule comprises:    -   a. at least one first domain comprising at least a portion of a        microbe-binding domain of a microbe-binding protein, wherein the        microbe-binding protein is not CRP;    -   b. at least one second domain comprising at least a portion of a        domain selected from the group consisting of:        -   i. Fc region of an immunoglobulin;        -   ii. neck region of a lectin;        -   iii. a detectable label;        -   iv. domain for conjugation to surface of a carrier scaffold;            and        -   v. any combinations of (i)-(iv); and    -   c. a linker conjugating the first and second domains.-   81. The method of any of paragraphs 78-80, wherein the    anti-microbial agent is conjugated with the microbe-targeting    molecule.-   82. An assay for determining the presence or absence of a microbe in    a test sample, the assay comprising:    -   (i) contacting a test sample with a c-reactive protein or a        portion thereof; and    -   (ii) detecting binding of a microbe to the c-reactive protein,    -   wherein a microbe is present in the test sample if binding is        detected.-   83. The assay of paragraph 82, wherein the c-reactive protein is    wild-type protein.-   84. The assay of paragraph 82, wherein the c-reactive protein is a    recombinant protein.-   85. The assay of any of paragraphs 82-84, wherein the c-reactive    protein is conjugated with a carrier scaffold.-   86. The assay of any of paragraphs 82-85, wherein said detecting    step comprises an enzyme-linked immunosorbent assay (ELISA), a    fluorescent linked immunosorbent assay (FLISA), immunofluorescent    microscopy, hybridization, fluorescence in situ hybridization    (FISH), antibody-based imaging, a radiological detection assay, a    chemical detection assay, an enzymatic detection assay, an optical    detection assay, an electrochemical assay, cell culture, or any    combinations thereof.-   87. The assay of any of paragraphs 82-86, wherein said detecting    step comprises contacting the sample from step (i) with a c-reactive    protein conjugated with a detectable label.-   88. A nucleic acid encoding c-reactive protein, wherein the nucleic    acid comprises the nucleic acid sequence SEQ ID NO: 45, SEQ ID NO:    46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49, SEQ ID NO: 50, SEQ    ID NO: 51, or any combinations thereof.-   89. An expression vector comprising a nucleic acid SEQ ID NO: 45,    SEQ ID NO: 46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49, SEQ ID    NO: 50, SEQ ID NO: 51, or any combinations thereof.-   90. A method for synthesizing a peptide comprising at least a    portion of a CRP, the method comprises expressing a expression    vector in a cell, wherein the expression vector comprises a nucleic    acid sequence SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47; SEQ ID    NO: 48; SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or any    combinations thereof.-   91. An assay for determining the presence or absence of a microbe in    a test sample, the assay comprising:    -   (i) contacting a test sample with c-reactive protein or a        microbe binding portion thereof; and    -   (ii) detecting binding of a microbe to the c-reactive protein or        a microbe binding portion thereof,    -   wherein a microbe is present in the test sample if binding is        detected.-   92. The assay of paragraph 91, wherein said detecting step comprises    an enzyme-linked immunosorbent assay (ELISA), a fluorescent linked    immunosorbent assay (FLISA), immunofluorescent microscopy,    hybridization, fluorescence in situ hybridization (FISH),    antibody-based imaging, a radiological detection assay, a chemical    detection assay, an enzymatic detection assay, an optical detection    assay, an electrochemical assay, cell culture, or any combinations    thereof.-   93. The assay of any of paragraphs 91 or 92, wherein said detecting    step comprises contacting the sample from step (i) with a    microbe-targeting molecule of any of paragraphs 1-29, wherein the    microbe-targeting molecule is conjugated with a detectable label.-   94. The assay of any of paragraphs 91-93, wherein the contacting    step comprises flowing the test sample through a channel, wherein    the channel is coated with the microbe-targeting molecules.-   95. The assay of any of paragraphs 91-94, wherein the contacting    step comprises flowing the test sample and microbe-targeting    molecule through a channel.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments of the aspects described herein, andare not intended to limit the claimed invention, because the scope ofthe invention is limited only by the claims. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Thus for example, references to “the method” includes one ormore methods, and/or steps of the type described herein and/or whichwill become apparent to those persons skilled in the art upon readingthis disclosure and so forth.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “microbe-binding” and “microbe-targeting” as usedinterchangeably herein refers to an ability of a molecule or compositionto bind and/or capture a microbe and/or microbial matter.

The term “FcCRP microparticle” as used herein refers to a microparticlecomprising on its surface at least one FcCRP molecule. In someembodiments, the microparticle comprises on its surface a saturatingamount of the FcCRP molecules. A microbead can be magnetic ornon-magnetic.

The term “FcCRPmagnetic microparticle” as used herein refers to amagnetic microbead comprising on its surface at least one FcCRPmolecule. In some embodiments, the magnetic microparticle comprises onits surface a saturating amount of the FcCRP molecules.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules(molecules that contain an antigen binding site which specifically bindsan antigen), including monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(for example, bispecific antibodies), chimeric antibodies, humanizedantibodies, human antibodies, and single chain antibodies (scFvs).

The term “peptide” refers to a polymer of amino acids, or amino acidanalogs, regardless of its size or function. In some embodiments, theterm “peptide” refers to small polypeptides, e.g., a polymer of about15-25 amino acids.

The term “oligonucleotide” as used herein refers to a short nucleic acidpolymer, typically with twenty or fewer bases.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein.

In some embodiments, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disease or disorder caused byany microbes or pathogens described herein. By way of example only, asubject can be diagnosed with sepsis, inflammatory diseases, orinfections.

The term “therapeutic agents” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. Various forms of atherapeutic agent may be used which are capable of being released fromthe subject composition into adjacent tissues or fluids uponadministration to a subject. Examples include steroids and esters ofsteroids (e.g., estrogen, progesterone, testosterone, androsterone,cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid,and chenodeoxycholic acid), boron-containing compounds (e.g.,carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,antivirals, antifungals), enediynes (e.g., calicheamicins, esperamicins,dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore),heavy metal complexes (e.g., cisplatin), hormone antagonists (e.g.,tamoxifen), non-specific (non-antibody) proteins (e.g., sugaroligomers), oligonucleotides (e.g., antisense oligonucleotides that bindto a target nucleic acid sequence (e.g., mRNA sequence)), peptides,proteins, antibodies, photodynamic agents (e.g., rhodamine 123),radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89,Ho-166, Sm-153, Cu-67 and Cu-64), toxins (e.g., ricin), andtranscription-based pharmaceuticals.

As used here in, the term “peptidomimetic” means a peptide-like moleculethat has the activity of the peptide on which it is structurally based.Such peptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,and have an activity such as the cardiac specificity of the peptide uponwhich the peptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery”, Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861).

A variety of peptidomimetics are known in the art and can be encompassedwithin embodiments described herein including, for example, peptide-likemolecules which contain a constrained amino acid, a non-peptidecomponent that mimics peptide secondary structure, or an amide bondisostere. A peptidomimetic that contains a constrained, non-naturallyoccurring amino acid can include, for example, an α-methylated aminoacid; α,α-dialkylglycine or α-aminocycloalkane carboxylic acid; anNα-Cαcyclized amino acid; an Nα-methylated amino acid; αβ- or γ-aminocycloalkane carboxylic acid; an α,β-unsaturated amino acid; aβ,β-dimethyl or β-methyl amino acid; αβ-substituted-2,3-methano aminoacid; an N—Cδor Cα-Cδcyclized amino acid; a substituted proline oranother amino acid mimetic. A peptidomimetic which mimics peptidesecondary structure can contain, for example, a nonpeptidic β-turnmimic; γ-turn mimic; mimic of β-sheet structure; or mimic of helicalstructure, each of which is well known in the art. A peptidomimetic alsocan be a peptide-like molecule which contains, for example, an amidebond isostere such as a retro-inverso modification; reduced amide bond;methylenethioether or methylene-sulfoxide bond; methylene ether bond;ethylene bond; thioamide bond; transolefin or fluoroolefin bond;1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylenebond or another amide isostere. One skilled in the art understands thatthese and other peptidomimetics are encompassed within the meaning ofthe term “peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptidedescribed herein, as well as potential geometrical and chemicalcomplementarity to a cognate receptor. Where no crystal structure of apeptide described herein is available, a structure can be generatedusing, for example, the program CONCORD (Rusinko et al., J. Chem. Inf.Comput. Sci. 29:251 (1989)). Another database, the Available ChemicalsDirectory (Molecular Design Limited, Informations Systems; San LeandroCalif.), contains about 100,000 compounds that are commerciallyavailable and also can be searched to identify potential peptidomimeticsof a peptide described herein, for example, having specificity for themicrobes.

The terms “homology” as used herein refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base or amino acid, then themolecules are identical at that position; when the equivalent siteoccupied by the same or a similar amino acid residue (e.g. , similar insteric and/or electronic nature), then the molecules can be referred toas homologous (similar) at that position. Expression as a percentage ofhomology refers to a function of the number of identical or similaramino acids at positions shared by the compared sequences. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity.Determination of homologs of the genes or peptides described herein maybe easily ascertained by the skilled artisan.

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the polypeptide's activity, fore examples,a conservative substitution refers to substituting an amino acid residuefor a different amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine. “Conservative amino acid substitutions” resultfrom replacing one amino acid with another having similar structuraland/or chemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine. Thus, a “conservative substitution” of a particular amino acidsequence refers to substitution of those amino acids that are notcritical for polypeptide activity or substitution of amino acids withother amino acids having similar properties (e.g., acidic, basic,positively or negatively charged, polar or non-polar, etc.) such thatthe substitution of even critical amino acids does not substantiallyalter activity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984).) In addition, individual substitutions, deletions or additionsthat alter, add or delete a single amino acid or a small percentage ofamino acids in an encoded sequence are also “conservativesubstitutions.” Insertions or deletions are typically in the range ofabout 1 to 5 amino acids.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

As used herein, “genetic elements” refers to defined nucleic acids(generally DNA or RNA) having expressible coding sequences for productssuch as proteins, apoproteins, or antisense nucleic acid constructs,which can perform or control pathway enzymatic functions. The expressedproteins can function as enzymes, repress or depress enzyme activity, orcontrol expression of enzymes. The nucleic acids encoding theseexpressible sequences can be either chromosomal, e.g. integrated into anonhuman organism's chromosome by homologous recombination,transposition, or some other method, or extrachromosomal (episomal),e.g. carried by plasmids, cosmids, etc. Genetic elements include controlelements. Many other genetic elements are known in the art. See, forexample, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,759,828; 5,888,783 and,5,919,670.

As used herein, the term “genetic manipulation” refers to the purposefulalteration of polynucleotide sequences either by in vitro techniques, invivo techniques, or a combination of both in vitro and in vivotechniques. “Genetic manipulation” includes the introduction ofheterologous polynucleotide sequences into nonhuman organisms, eitherinto the chromosome or as extrachromosomaily replicating elements, thealteration of chromosomal polynucleotide sequences, the addition and/orreplacement of transcriptional and/or translational regulatory signalsto chromosomal or plasmid encoded genes, and the introduction of variousinsertion, deletion and replacement mutations in genes of interest.Methods for in vitro and in vivo genetic manipulations are widely knownto those skilled in the art. See, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborPress (1989) and U.S. Pat. Nos. 4,980,285; 5,631,150; 5,759,828;5,888,783 and, 5,919,670.

As used herein, “operably linked” refers to juxtaposition such that thenormal function of the components can be performed. Thus, a codingsequence “operably linked” to control sequences refers to aconfiguration wherein the coding sequences can be expressed under thecontrol of these sequences. Such control may be direct, that is, asingle gene associated with a single promoter, or indirect, as in thecase where a polycistronic transcript is expressed from a singlepromoter. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150;5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborPress (1989).

As used herein, “over-expression” refers to gene expression. Genes andgene products can be overexpressed. Such gene products include RNAs,proteins and enzymes. On the other hand, “overproduce” refers tocellular products that accumulate, especially cell products that are tobe harvested for some specific use. Thus proteins, materials (such aspolymers), and metabolites (such as amino acids) are overproduced.Proteins may be either overexpressed (if referring to the control ofgene expression) or overproduced (if referring to the accumulation ofthe proteins). By “over production” of ergothioneine, it is intendedthat a cell “overproducing” ergothioneine produces more molecules ofergothioneine for each cell under a given set of growth conditions thana similar cell not “over producing” ergothioneine.

As used herein, the term “promoter” has its art-recognized meaning,denoting a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes. Sequence elements within promoters that function inthe initiation of transcription are often characterized by consensusnucleotide sequences. Useful promoters include constitutive andinducible promoters. Many such promoter sequences are known in the art.See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828;5,759,828; 5,888,783; 5,919,670, and, Sambrook et al, Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). Otheruseful promoters include promoters which are neither constitutive norresponsive to a specific (or known) inducer molecule. Such promoters mayinclude those that respond to developmental cues (such as growth phaseof the culture or stage of cell differentiation), or environmental cues(such as pH, osmoticum, heat, or cell density). A heterologous promoteris a promoter which is not naturally linked to the gene. Heterologouspromoters may be from the same or different species. For example, aheterologous promoter may be a promoter from the same organism as thegene but naturally found linked to a different gene.

As used herein, the term “transgene” when used in reference topolynucleotide sequences, refers to polynucleotide sequences notnaturally present in a cell. Thus the term “transgene” includes, forexample, the promoter of gene A operably joined to structural gene B,when A and B genes are from the same organism, as well as the case inwhich a polynucleotide sequence of one species is transferred to a cellof a different species (or strain). The term “transgene” also includesclones of transgenes which have been so modified. See, U.S. Pat. Nos.4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670.

As used herein, the terms “culture media,” and “cell culture media,”refers to media that are suitable to support the growth of cells invitro (i.e., cell cultures). It is not intended that the term be limitedto any particular cell culture medium. For example, it is intended thatthe definition encompass outgrowth as well as maintenance media. Indeed,it is intended that the term encompass any culture medium suitable forthe growth of the cell cultures of interest.

As used herein, the term “cell type,” refers to any cell, regardless ofits source or characteristics.

As used herein, the term “transformed cell lines,” refers to cellcultures that have been transformed into continuous cell lines with thecharacteristics as described herein.

As used herein, the term “transformed nonhuman organisms” includes theprimary transformed subject cell and its transformed progeny. Thenonhuman organism can be prokaryotic or eukaryotic. Thus “transformants”or “transformed cells” includes the primary subject cell, transformedwith the transgene, and cultures derived therefrom, without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations and/or modifications. Mutant progeny which have the samefunctionality as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150;5,707,828; 5,759,828; 5,888,783; 5,919,670, and, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., 60 Cold Spring HarborPress (1989).

As used herein, the term “isolated” means altered “by the hand of man”from the natural state. An “isolated” composition or substance is onethat has been changed or removed from its original environment, or both.For example, a polynucleotide or a polypeptide naturally present in acell or living animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated,” as the term is employed herein.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedmay be further modified to incorporate features shown in any of theother embodiments disclosed herein.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such canvary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1: C-Reactive Protein Capture/Detection of GRAM POSITIVEBacteria

C-reactive protein (CRP) is an acute inflammation protein originallycharacterized by its ability to bind to C-polysaccharide from the cellwall of Streptococcus pneumoniae (1). CRP helps bridge the innate andadaptive immune responses primarily through activation of complement,opsonization, and induction of phagocytosis (2). As a two-sidedallosteric pentamer, CRP functions by binding to host cell proteins onone side and phosphocholine-containing debris from pathogenic microbeson the other in calcium-dependent zwitterionic interactions (2, 3).Despite CRP's original characterization as a Pattern RecognitionReceptor, its primary use in medicine is as a biomarker of systemicinflammation in critically ill patients, as well as a predictor ofatherosclerosis (4, 5, 6).

Using the ability of CRP to bind immunologically active components ofbacteria, we have developed an assay using CRP as both a capture anddetection agent for Enterococcus faecalis, Enterococcus faecium, andStreptococcus pneumoniae and other gram positive bacteria. Using thisassay, we have developed a Rapid assay with which we can detect andclassify Gram Positive bacteria, even where there is no positive bloodculture. This information can be used for optimizing antibiotictreatment.

Using the binding properties of CRP (coupled to superparamagnetic beads)we have developed an assay (CRP-ELISA) that can detect gram positivebacteria (Table 1) from complex media such as blood and can be used as arapid gram test for infected clinical samples. In addition, the CRPcoupled to either superparamagnetic beads or dialysis filters can beused to clear the pathogens from infected blood.

The current panel of pathogens tested to date demonstrate CRP coupledbeads are specific to gram positive bacteria. All gram negative bacteriatested are negative for binding. Therefore the use of CRP to capture anddetect bacteria (gram positive) can be used as a rapid gram test ofinfected patient samples.

An exemplary detection assay using the microbe-trageting moleculesaccording to some embodiments:

-   -   1. Bead: CRP is coupled to superparamagnetic beads (example: CRP        is biotinylated and coupled to streptavidin coated 1 uM MyONE        beads from ThermoFisher).    -   2. Capture: CRP beads are added to the test sample (example        infected blood), mixed (capture step), removed, and washed for        assaying. Optimum time and shake speed to be determined.    -   3. Detection: Bead/pathogen complex is assayed by CRP-HRP ELISA        (HRP=horseradish peroxidase) (FIG. 5). Optimum assay conditions        to be determined, for example incubation time, CRP-HRP        concentration etc.    -   4. Sample protocol: Bead/pathogen mixes are incubated with        CRP-HRP (1:2500 in TBST-Ca⁺⁺ 5 mM 3% BSA) for 20 minutes. Beads        are washed 3× in TBST Ca⁺⁺ 5 mM, 1× in TBS Ca⁺⁺ 5 mM and        positive capture by beads detected using colorimeteric detection        (TMB substrate).

TABLE 1 List of bacteria tested for CRP binding CRP Capture/DetectionAcinetobacter baumannii Yes Aeromonas sobria Yes Burkholderia cepaciaYes Citrobacter freundii Yes Enterobacter aerogenes Yes Enterobactercloacae Yes Enterococcus faecalis Yes Enterococcus faecium YesEscherichia coli Yes Klebsiella oxytoca Yes Klebsiella pneumoniae YesListeria monocytogenes Yes Pseudomonas aeruginosa Yes Salmonellaenteriditis Yes Salmonella typhimurium Yes Serratia marcescens YesStaphylococcus aureus Yes Staphylococcus aureus (MRSA) YesStaphylococcus epidermidis Yes Staphylococcus lugdunensis YesStreptococcus agalactiae Yes Streptococcus Group A Yes Streptococcusmitis Yes Streptococcus pneumoniae Yes Streptococcus pyogenes YesCapture/binding of clinical isolates was determined by CRP ELISA using 1μM CRP coated beads and detection by CRP-HRP

REFERENCES

-   1. Tillet W S, Francis T. Serological reactions in pneumonia with a    non-protein somatic fraction of the Pneumococcus. J Exp Med 1930;    52: 561-71.-   2. Szalai, A I. The biological functions of C-reactive protein. Vasc    Pharm 2002; 39: 105-107.-   3. Barnum et al. Comparative studies on the binding specificities of    C-reactive Protein and HOPC. Annals of NYAS 1982; 4: 431-434.-   4. Tsalik et al. Discriminative value of inflammatory biomarkers of    sepsis. J Emerg Med 2012; 43: 1.97-106.-   5. Levy M M, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International    Sepsis Definitions Conference. Crit Care Med 2003; 31: 1250-6.-   6. Ablij H C, Meinders A E. C-reactive protein: history and revival.    EJIM 2002; 13: 412-422.

C-reactive protein's ability to bind to the phosphorylcholine moietieson the surface of pathogenic bacteria may be useful in the diagnosis andcharacterization of infection and sepsis. Early research demonstratedthe importance of C-reactive protein in the innate immune response tomicrobial infection, but much of contemporary research on C-reactiveprotein (CRP) has focused on its usefulness as a biomarker ofinflammation and predictor of cardiovascular disease. This studyrevisits the function of CRP as a binder of immunologically activemolecules and its function in innate immune defense. Using CRP-coatedmagnetic microbeads, intact Enterococcus faecalis and Enterococcusfaecium were bound and removed by magnet from a serum-like buffersolution. The total concentration of bacteria dropped from 10,000 CFU/mLto 1000 CFU/mL, a significant reduction when compared with controls.Using a magnetic bead-based ELISA with CRP as the detection agent,intact Enterococci were detected at concentrations as low as 100 CFU/mL.When run with mechanically fragmented bacterial cells, sensitivedetection of Enterocci, Streptococci and Staphylococci was possible.Detection of fragments in buffer has clinical relevance because itmimics the physiologically conditions of infection. Fragments of deadcells far outnumber living bacteria in cases of sepsis. CRP's ability tobind pathogenic bacterial fragments is relevant in the diagnostics forsepsis.

Example 2: Enhanced CRP Expression

Genes for the following proteins were cloned into a mammalian expressionvector downstream of a retroviral promoter. This vector was transfectedinto HEK 293F cells using 293Fectin. Supernatents from the transfectedcells was harvested after cell viability dropped below 50% and CRPcontaining proteins were bound to on immobilized p-AminophenylPhosphoryl Choline in the presense of calcium and eluted using an EDTAcontaining buffer.

TABLE 2 Encoded Amino Yield DNA Acid (mg Sequence Sequence Protein/ (SEQID (SEQ ID L cell Name NO:) NO:) Design Rationale culture) wtCRP 52 39none wild type 0.3 cDNA OPTCRP 45 39 Heuristically optimized 35 sequenceOptCRPv2b 46 39 WT sequence with just 4 Mixes between 0.3 codonsoptimized for human optimized and bias (Pro25, Arg47, Cys97, wt CRP DNACys36) sequence to identify minimal DNAs needed to achieve high CRPexpressions OptCRPv2c 47 39 WT sequence - all codons able Mixes between7 to be improved 2.5 fold in optimized and terms of human usage replacedwt CRP DNA sequence to identify minimal DNAs needed to achieve high CRPexpressions OptCRPv2d 48 39 1-97 WT, 98-end optimized Mixes between 5sequence optimized and wt CRP DNA sequence to identify minimal DNAsneeded to achieve high CRP expressions OptCRPv2e 49 39 1-97 optimized,98-end WT Mixes between 14 optimized and wt CRP DNA sequence to identifyminimal DNAs needed to achieve high CRP expressions OptCRPv2F 50 39 1-52& 170-end WT; middle Mixes between 5 optimized sequence optimized and wtCRP DNA sequence to identify minimal DNAs needed to achieve high CRPexpressions OptCRPv2g 51 39 1-52, 170-end optimized; Mixes between 14middle wild-type sequence optimized and wt CRP DNA sequence to identifyminimal DNAs needed to achieve high CRP expressions

SEQUENCES:HumanCRP (NCBI Reference Sequence: NP_000558.2, (SEQ ID NO: 1)MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLEIFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWPMouseCRP (NCBI Reference Sequence: NP_031794.3, SEQ ID NO: 2)MEKLLWCLLIMISFSRTFGHEDMFKKAFVFPKESDTSYVSLEAESKKPLNTFTVCLHFYTALSTVRSFSVFSYATKKNSNDILIFWNKDKQYTFGVGGAEVRFMVSEIPEAPTHICASWESATGIVEFWIDGKPKVRKSLEIKGYTVGPDASIILGQEQDSYGGDFDAKQSLVGDIGDVNMWDFVLSPEQISTVYVGGTLSPNVLNWRALNYKAQGDVFIKPQLWS CRP sequence(SEQ ID NO: 3)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLEIFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTK CRP Sequence with Cys to Ala modifications -(SEQ ID NO: 4)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVALHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHIATSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP Fc Sequence  (SEQ ID NO: 5)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAFc Sequence with Asn82Asp modification  (SEQ ID NO: 6)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Fc-neck sequence(SEQ ID NO: 7)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPDGDSSLAASERKALQTEMARIKKWLTFSLG MBL full length (SEQ ID NO: 8):MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPC STSHLAVCEFPIMBL without the signal sequence  (SEQ ID NO: 9)ETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI MBL signal sequence (SEQ ID NO: 10) MSLFPSLPLLLLSMVAASYS Truncated MBL  (SEQ ID NO: 11)AASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI Carbohydrate recognition domain (CRD) of MBL (SEQ ID NO: 12)VGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPINeck + Carbohydrate recognition domain of MBL (SEQ ID NO: 23)PDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI FcMBL.81  (SEQ ID NO: 24)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI AKT-FcMBL (SEQ ID NO: 25)AKTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI FcMBL.111  (SEQ ID NO: 26)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGATSKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI CRD Sequence(SEQ ID NO: 27)KQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIFcCRP, where Fc is the Fragment crystallization from IgG and CRP is themature form of CRP(SEQ ID NO: 33)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG<linker>QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLEIFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTK (<1inker> refers of a linker from 0 to 20 amino acids, e.g., 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector)FcmCRP, where mCRP refers to a monomeric version of mature CRP (SEQ ID NO: 34)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG<linker>QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVALHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHIATSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP(<1inker> refers of a linker from 0 to 20 amino acids, e.g.,0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector)CRP-CRD, where CRD refers to any carbohydrate binding domain from a C-type lectin, e.g.,MBL (SEQ ID NO: 35)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLEIFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP<linker>KQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI (<1inker> refers of a linker from 0 to 20 aminoacids, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector CRD-CRP(SEQ ID NO: 36)KQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI<linker>QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLEIFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP (<1inker> refers of a linker from 0 to 20 aminoacids, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector)Fc-neck-mCRP, where neck refers to the tnple helix neck from a Collectins, e.g., MBL (SEQ ID NO: 37)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPDGDSSLAASERKALQTEMARIKKWLTFSLG<linker>QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVALHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHIATSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP(<linker> refers of a linker from 0 to 20 amino acids, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector)AKT-His-CRP; where AKT is a tri-peptide for site specific conjugation of the protein and Hisrefers to a histidine tag for purification  (SEQ ID NO: 38)AKTHREIHHHQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP wtCRP (SEQ ID NO: 39)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP CRP-Fc; (N297D variant) with/withouta linker (SEQ ID NO: 40)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP<linker>EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA (<1inker> refers of a linker from 0 to 20 amino acids,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector)CRP-Fc; (N297D variant) with a single leucine residue as a linker (SEQ ID NO: 41)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWPLEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA Fc sequence; N297D variant  (SEQ ID NO: 42)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGACRP-Fc; N297 with without a linker  (SEQ ID NO: 43)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP<linker>EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA (<1inker> refers of a linker from 0 to 20 amino acids,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the linker can be encoded in the expression vector) CRP-Fc; N297 no linker (SEQ ID NO: 44)QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA cDNA; heuristically optimized sequence (SEQ ID NO: 45)CAGACCGATATGAGCCGGAAAGCCTTCGTGTTCCCTAAAGAGAGCGATACCAGCTATGTGAGCCTGAAAGCCCCACTGACCAAACCACTGAAGGCCTTCACCGTGTGCCTGCACTTTTACACCGAGCTGAGCTCCACACGAGGGTACAGCATCTTTTCCTATGCTACAAAGAGGCAGGACAACGAAATCCTGATTTTCTGGTCAAAAGATATCGGCTATAGCTTTACTGTGGGCGGATCCGAGATTCTGTTCGAGGTGCCCGAAGTCACAGTGGCTCCTGTCCATATCTGTACTTCCTGGGAGTCAGCAAGCGGCATTGTCGAATTCTGGGTGGACGGAAAGCCTAGGGTGAGAAAATCTCTGAAGAAAGGATACACAGTGGGGGCCGAGGCTAGTATCATTCTGGGGCAGGAACAGGACTCATTCGGGGGCAACTTTGAAGGCTCCCAGTCTCTGGTCGGGGATATCGGCAACGTGAACATGTGGGACTTTGTCCTGAGCCCAGATGAGATCAATACCATCTACCTGGGAGGGCCCTTCAGCCCTAACGTGCTGAATTGGCGCGCACTGAAGTATGAGGTCCAGGGCGAAGTGTTTACAAAACCCCAGCTGTGGCCAcDNA; WT sequence with just 4 codons optimized for human bias (Pro25, Arg47, Cys97, Cys36);CRP v2b  ((SEQ ID NO: 46)CAGACAGACATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCCTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCAGAGGGTACAGTATTTTCTCGTATGCTACTAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGCACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCCcDNA; WT sequence - all codons able to be improved 2.5 fold in terms of human usage replaced;CRP v2c  (SEQ ID NO: 47)CAGACAGACATGAGCAGGAAGGCTTTTGTGTTTCCCAAAGAGAGCGATACTTCCTATGTGTCCCTCAAAGCACCGCTGACCAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACCGAACTGTCCAGCACCCGTGGGTACAGTATTTTCAGCTATGCTACTAAGAGACAGGACAATGAGATTCTCATATTTTGGTCTAAGGATATCGGATACAGTTTTACAGTGGGTGGGTCTGAAATACTGTTCGAGGTGCCTGAAGTCACAGTGGCTCCAGTACACATTTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTGGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCCTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTGGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAGGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCCcDNA; 1-97 WT, 98-end optimized sequence; CRP v2d  (SEQ ID NO: 48)CAGACAGACATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCGTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCGTATGCTACTAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGTACTTCCTGGGAGTCAGCAAGCGGCATTGTCGAATTCTGGGTGGACGGAAAGCCTAGGGTGAGAAAATCTCTGAAGAAAGGATACACAGTGGGGGCCGAGGCTAGTATCATTCTGGGGCAGGAACAGGACTCATTCGGGGGCAACTTTGAAGGCTCCCAGTCTCTGGTCGGGGATATCGGCAACGTGAACATGTGGGACTTTGTCCTGAGCCCAGATGAGATCAATACCATCTACCTGGGAGGGCCCTTCAGCCCTAACGTGCTGAATTGGCGCGCACTGAAGTATGAGGTCCAGGGCGAAGTGTTTACAAAACCCCAGCTGTGGCCAcDNA; 1-97 optimized, 98-end WT; CRP v2e  (SEQ ID NO: 49)CAGACCGATATGAGCCGGAAAGCCTTCGTGTTCCCTAAAGAGAGCGATACCAGCTATGTGAGCCTGAAAGCCCCACTGACCAAACCACTGAAGGCCTTCACCGTGTGCCTGCACTTTTACACCGAGCTGAGCTCCACACGAGGGTACAGCATCTTTTCCTATGCTACAAAGAGGCAGGACAACGAAATCCTGATTTTCTGGTCAAAAGATATCGGCTATAGCTTTACTGTGGGCGGATCCGAGATTCTGTTCGAGGTGCCCGAAGTCACAGTGGCTCCTGTCCATATCTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCCcDNA; 1-52 & 170-end WT, middle optimized sequence; CRP v2f (SEQ ID NO: 50)CAGACCGATATGAGCCGGAAAGCCTTCGTGTTCCCTAAAGAGAGCGATACCAGCTATGTGAGCCTGAAAGCCCCACTGACCAAACCACTGAAGGCCTTCACCGTGTGCCTGCACTTTTACACCGAGCTGAGCTCCACACGAGGGTACAGCATCTTTTCGTATGCTACTAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATCAATACCATCTACCTGGGAGGGCCCTTCAGCCCTAACGTGCTGAATTGGCGCGCACTGAAGTATGAGGTCCAGGGCGAAGTGTTTACAAAACCCCAGCTGTGGCCAcDNA; 1-52, 170-end optimized, middle wild-type sequence; CRP v2g (SEQ ID NO: 51)CAGACAGACATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCGTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCCTATGCTACAAAGAGGCAGGACAACGAAATCCTGATTTTCTGGTCAAAAGATATCGGCTATAGCTTTACTGTGGGCGGATCCGAGATTCTGTTCGAGGTGCCCGAAGTCACAGTGGCTCCTGTCCATATCTGTACTTCCTGGGAGTCAGCAAGCGGCATTGTCGAATTCTGGGTGGACGGAAAGCCTAGGGTGAGAAAATCTCTGAAGAAAGGATACACAGTGGGGGCCGAGGCTAGTATCATTCTGGGGCAGGAACAGGACTCATTCAACTTTGAAGGCTCCCAGTCTCTGGTCGGGGATATCGGCAACGTGAACATGTGGGACTTTGTCCTGAGCCCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCC cDNA; wild type (SEQ ID NO: 52)CAGACAGACATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCGTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCGTATGCTACTAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCC

What is claimed is:
 1. A method for determining the presence or absenceof a microbe in a sample, comprising: (i) contacting a test sample withthe microbe-targeting molecule; and (ii) detecting binding of a microbeto the microbe-targeting molecule, wherein the microbe is present in thetest sample if binding is detected and wherein the microbe-targetingmolecule comprises: a. at least one first domain comprising at least aportion of a c-reactive protein (CRP); b. at least one second domaincomprising at least a portion of a domain selected from the groupconsisting of: (i) Fc region of an immunoglobulin; (ii) microbe-bindingdomain of a microbe-binding protein, wherein the microbe-binding proteinis not CRP; (iii) neck region of a lectin; (iv) a detectable label; (v)domain for conjugation to surface of a carrier scaffold; (vi) patternrecognition receptor domain of CRP; and (vii) any combinations of(i)-(vi); and c. a linker conjugating the first and second domains, andwherein the microbe-targeting molecule is a multimeric molecule.
 2. Themethod of claim 1, wherein said detecting step comprises anenzyme-linked immunosorbent assay (ELISA), a fluorescent linkedimmunosorbent assay (FLISA), immunofluorescent microscopy,hybridization, fluorescence in situ hybridization (FISH), antibody-basedimaging, a radiological detection assay, a chemical detection assay, anenzymatic detection assay, an optical detection assay, anelectrochemical assay, cell culture, or any combinations thereof.
 3. Themethod of claim 1, wherein the microbe-targeting molecule is conjugatedwith a detectable label.
 4. The method of claim 1, wherein themicrobe-bound microbe-targeting molecule is detectable without priorcell culture.
 5. The method of claim 1, wherein the contacting stepcomprises flowing the test sample through a channel, wherein the channelis coated with the microbe-targeting molecules.
 6. The method of claim1, wherein the contacting step comprises flowing the test sample andmicrobe-targeting molecule through a channel.
 7. The method of claim 1,wherein said detecting in step (ii) comprises contacting the sample witha labeling molecule conjugated with a detectable label.
 8. The method ofclaim 1, wherein the multimeric molecule is formed by: interactionsbetween the linkers of different molecules forming the multimericmolecule; or the linker and the second domain of different moleculesforming the multimeric molecule; or the second domains of differentmolecules forming the multimeric molecule.
 9. The method of claim 1,wherein the Fc region comprises at least one mutation.
 10. The method ofclaim 1, wherein the microbe-binding protein is a carbohydrate bindingprotein.
 11. The method of claim 10, wherein the microbe-binding domainis a carbohydrate recognition domain (CRD) of the carbohydrate bindingprotein.
 12. The method of claim 11, wherein the CRD is from a lectin orficolin.
 13. The method of claim 1, wherein the second domain comprisesat least a portion of Fc region of an immunoglobulin and at least aportion of the neck region of a lectin.
 14. The method of claim 1,wherein the detectable molecule is selected from the group consisting ofbiotin, fluorophore, luminescent or bioluminescent marker, a radiolabel,an enzyme, an enzyme substrate, a quantum dot, an imaging agent, a goldparticle, and any combinations thereof.
 15. The method of claim 1,wherein the domain for conjugation to surface of a carrier scaffoldcomprises an amino group, a N-substituted amino group, a carboxyl group,a carbonyl group, an acid anhydride group, an aldehyde group, a hydroxylgroup, an epoxy group, a thiol, a disulfide group, an alkenyl group, ahydrazine group, a hydrazide group, a semicarbazide group, athiosemicarbazide group, one partner of a binding pair, an amide group,an aryl group, an ester group, an ether group, a glycidyl group, a halogroup, a hydride group, an isocyanate group, an urea group, or anurethane group.
 16. The method of claim 1, wherein the microbe-targetingmolecule is conjugated to a surface of a carrier scaffold.